Progress 02/01/10 to 01/31/14
Outputs
Target Audience: Target audiences that have been reached by this project: 1. Two research publications and one review article are primarily for the benefit of other scientists. However, all of these articles are available on the web and can be accessed by the general public. The review article, in particular, may be of interest to non-scientists. 2. Undergraduate students in a class taught by co-PD Wilson have made use of Myzus persicae genome sequence data that were generated through this project. 3. The Myzus persicae genome sequence data have been made publicly available through AphidBase (www.aphidbase.org), which is maintained by the French collaborators for this project, Denis Tagu and Fabrice Legai. The target audience is anyone who has an interest in the M. persicae genome sequence. To date, 75 unique users have downloaded sequences from this database. Changes/Problems: Fewer full-length genes encoding secreted salivary proteins were available than had been originally predicted in the grant proposal. Therefore, a smaller number of genes was investigated in more detail. In particular, aphid gene silencing approaches, which were not available at the time of writing the proposal, were used to confirm gene function. As the price of DNA sequencing decreased during the course of the project, more sequencing was accomplished than had been originally proposed. Publication of the genome sequencing results has been delayed due to the necessity of coordinating with the group of Saskia Hogenhout at the John Innes Center, which has sequences a different M. persicae lineage. However, we believe that this delay will result in a better publication with two M. persicae genomes that can be compared. What opportunities for training and professional development has the project provided? One postdoc, two graduate students, and two undergraduates received training through this project. Daniel Price (postdoc) received training in RNA extractions for RNAseq library preparation Dezi Elzinga (graduate student) received training in molecular biology, gene cloning, RNAseq, aphid bioassays, and manuscript writing. Honglin Feng (graduate student) receive training in aphid rearing, tissue dissection, and preparation of RNAseq libraries. Elizabeth Isner and Paul Enger (undergraduates) learned bioinformatics methods. How have the results been disseminated to communities of interest? 1. Three manuscripts have been published in scientific journals. 2. The four Buchnera apidicola genomes are in GenBank. 3. Myzus persicae genome sequences are in AphidBase, www.aphidbase.org 4. Two additional manuscripts describing the salivary gland transcriptomes and the M. persicae genome are in preparation and will cite funding from this grant. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project had two main goals: 1) functional analysis of aphid salivary proteins, and 2) sequencing of the Myzus persicae genome. 1. Functional analysis of aphid salivary proteins. Based on published proteomic data of secreted aphid salivary proteins, Dezi Elzinga, a graduate student in the Jander lab, amplified M. persicae genes and cloned them into plant expression vectors. Based on success in obtaining full-length genes and effects of cloned genes in plants, eight aphid salivary genes were chosen for more detailed analysis. These genes were expressed in tobacco and Arabidopsis to measure their effects on aphid reproduction. A virus-induced gene silencing approach was used to reduce expression of salivary genes in aphids feeding on tobacco. Agrobacterium-mediated expression of double-stranded RNA was used to silence expression of salivary genes in aphids feeding from Arabidopsis. Overexpression of salivary genes in plants and silencing of these genes in aphids had opposite effects on aphid reproduction. These results were published in the journal Molecular Plant-Microbe Interactions. In an alternate approach to studying aphid salivary genes, Dezi Elzinga performe transcript profiling (RNASeq) on aphid salivary glands extracted from aphids feeding on tobacco, cabbage, and Arabidopsis to identify host plant specific gene expression. Separate profiling was performed with the primary and accessory salivary glands. In parallel, James Carolan (University College Dublin) performed a proteomic analysis of salivary glands harvested in parallel. These results are being prepared for publication in a journal and inclusino in Elzinga's PhD thesis, which will be defended in the summer of 2014. 2. Myzus persicae genome sequencing In a project directed by Alex Wilson, a draft sequence of the M. persicae genome (Clone G006, collected in Geneva, New York) was obtained. Due to a rapid drop in the price of sequencing, a greater coverage of the genome was obtained than had additionally been proposed. Additionally, deep transcriptome sequencing was performed to facilitate annotation of the expressed genes. Collaborators at INRA in France, who were funded through this project, a BLAST-searchable database of the M. persicae genome sequence was set up (www.aphidbase. org). In the course of this project, we learned that the group of Saskia Hogenhout at the John Innes Center was also seqeuncing an M. persicae genome (Clone O, found throughout Britain). We are coordinating the the Hogenhout group to annotate both genomes in the same manner. . To date, ~16,000 genes have been identified in the two aphid lineages. There is a 90% overlap in the sets of genes found in Clone G006 and Clone O, respectively. The genome browser at AphidBase is being adapted to allow community annotation of M. persicae genes. A joint publication of the G006 and Clone O genomes will be published when the assembly and gene annotation are sufficiently complete. As part of sequencing the M. persicae genome, Wilson and co-workers also sequenced the genomes of four isolates of the Buchnera aphidicola endosymbiont of M. persicae. Two of these isolates were red tobacco-adapted lineages of M. persicae and two were green non-tobacco lineages. These are the first genome sequences of the bacterial endosymbiont bacteria of M. persicae. A paper comparing the four B. aphidicola Mp genome sequences to each other and to other published genomes of B. aphidicola endosybionts from other aphid species has been published in BMC Genomics.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Elzinga DA, De Vos M, and Jander G (2014) Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein, Molecular Plant-Microbe interactions, advance online publication.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Jiang Z, Jones DH, Khuri S, Tsinoremas NF, Wyss T, Jander G, Wilson ACC (2014) Comparative analysis of genome sequences from four strains of the Buchnera aphidicola Mp endosymbiont of the green peach aphid, Myzus persicae. BMC Genomics, 14, 917.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Elzinga DA and Jander G (2013)The role of protein effectors in plant-aphid interactions. Current Opinion in Plant Biology 16:451-456.
|
Progress 02/01/11 to 01/31/12
Outputs
Target Audience: Aphid researchers are the primary target audience for the most recent results from this project. Both the Myzus persicae genome sequence and new methods for gene silencing will accelerate research on this important agricultural pest. Myzus persicae chromosomal DNA sequences have been made available to the public and other scientists via the AphidBase website, www.aphidbase.com during this reporting period. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Students that are being trained through the project are Dezi Elzinga, Derek Jones, and Tania Wyss. Project participants are working at the Boyce Thompson Institute (Georg Jander and Dezi Elzinga) and the University of Miami (Alex Wilson, Zhijie Jiang, Derek Jones Sawsan Khuri, Nicholas Tsinoremas, and Tania Wyss). Collaborators are working at INRA (Denis Tagu and Fabrice Legai), Rothamsted Research (Saskia Hogenhout and Lin Field), the James Hutton Institute (Brian Fenton), and the National University of Ireland Maynooth (James Carolan). How have the results been disseminated to communities of interest? DNA sequences of Myzus persicae and its endosymbiont bacteria, Buchnera aphidicola, have been made available through GenBank and AphidBase. What do you plan to do during the next reporting period to accomplish the goals? 1. A publication describing the Buchnera endosymbiont genome of Myzus persicae will be published. 2. Further work will be done to characterize the salivary proteome of Myzus persicae and investigate the function of these proteins in plants. Aphid salivary proteins will be expressed in tobacco and Arabidopsis to study their function in plant defense interactions. 3. In collaboration with the lab of Saskia Hogenhout, Myzus persicae genomic sequences will be characterized and compared in preparation for publication.
Impacts
What was accomplished under these goals?
Twelve Myzus persicae salivary proteins have been functionally characterized by expression in plants. Of these, six affect plant resistance to aphid feeding. Two make the plants more aphid-sensitive and two make the plants more aphid-resistant. Similar effects are observed in Arabidopsis and tobacco. Initial experiments suggest that the aphid salivary proteins that make the plants more aphid-sensitive are suppressing plant defenses. A manuscript describing these results is being written. Two draft sequences of the Myzus persicae (green peach aphid) genome have been assembled in collaboration with researchers at Rothamsted Research in the United Kingdom and the French National Institute for Agricultural Research (INRA). Both sequences are publicly available for BLAST searches at AphidBase, http://tools.genouest.org/tools/myzus/login. The two sets of sequences are from two previously studied aphid lineages, a North American M. persicae strain, G006 (Ramsey et al, 2007, BMC Genomics 8:423 doi:10.1186/1471-2164-8-423), and a British strain, Clone O (Fenton et al, 2010, Ecological Entomology, 35:S131-S146 DOI: 10.1111/j.1365-2311.2009.01150.x), respectively. Once further assembly and corrections have been made, these genome sequences also will be submitted to GenBank. Genome assembly of the Buchnera aphidicola endosymbiont bacteria of M. persicae (Buchnera Mp) has been completed. Endosymbionts from four strains of M. persicae were sequenced, two from tobacco-adapted aphid lineages and two from non-tobacco-adapted lineages. The four bacterial genome sequences have been submitted to GenBank and also will be included in BuchneraBase (www.buchnera.org), a comparative database of insect bacterial endosymbiont genome sequences. Assembly of Buchnera aphidicola endosymbiont (Buchnera Mp) genomes from four lineages of M. persicae showed not differences in gene content. Two of these M. persicae lineages were tobacco adapted. Although some prior studies demonstrated that tobacco-adapted lineages are genetically distinct, there were no differences in the gene content of the Buchera Mp endosymbionts. Thus, it is unlikely that these bacteria contribute to the ability of some M. persicae strains to grow particularly well on tobacco. Comparisons to Buchnera from other aphid species suggest that certain genes in Buchnera Mp may facilitate feeding on a broad range of host plants. A manuscript describing these results has been submitted.
Publications
|
Progress 02/01/10 to 01/31/11
Outputs
OUTPUTS: Myzus persicae (green peach aphid) lineage G006 has a genome size of 410 Mb (J. Spencer Johnston unpublished data). Fifty mg, as estimated using Qubit quantitation, of genomic DNA for whole genome sequencing was prepared from large cultures of Myzus persicae lineage G006 using a DNAeasy DNA Extraction Kit (Qiagen Cat. No. 69504) and sent for Illumina library construction and sequencing at the University of Miami's Center for Genome Sequencing Core at the Hussman Institute for Human Genomics. One paired-end library size selected to 300-400 bp and two mate-paired libraries size selected to 2 kb and 5 kb were prepared and sequenced using a version 3 Illumina Hi-Seq paired-end flow cell. Illumina sequencing reads were assembled using 4 different assembler programs : Abyss (Simpson etal., 2009), Allpaths-LG (Gnerre et al., 2011), SOAPdenovo (R. Li et al., 2010) and Monument (Chikhi et al., 2012; an assembler developed by collaborators in the project team). The best assemblies were obtained using AllPaths-LG and Monument. In the case of Monument, assembly from 388,229,066 bp coverage, providing 9032 scaffolds, with a mean scaffold size of 877 bp. Sequence data are being shared with another group in England to create one combined M. persicae genome assembly. Main and accessory salivary glands were dissected from M. persicae for proteomic and transcriptomic experiments. Proteomic studies determined which proteins are in the salivary glands. Analysis of transcribed genes determines whether they are expressed in the main or accessory salivary glands. Analysis of M. persicae salivary gland gene expression on different host plants (Arabidopsis, cabbage, and tobacco) showed host-specific changes in expression. Genes for twenty M. persicae salivary proteins were expressed in Nicotiana benthamiana and/or Arabidopsis thaliana. In aphid bioassays six of these proteins affected aphid reproduction, with five decreasing and one increasing the number of progeny. Analysis of aphid salivary gland gene expression shows differences in response to expression of these proteins in the plants. Antibodies made to peptides contained in M. persicae salivary proteins will allow the in-planta detection of these proteins. PARTICIPANTS: Boyce Thompson Institute: Georg Jander has served as the principal investigator and coordinated the overall project. Dezi Elzinga, a graduate student, has conducted hands-on research and received training. University of Miami: Alex Wilson prepared samples for sequencing and analyzed data. INRA (France): Collaborators in the groups of Denis Tagu and Fabrice Legai assembled genome sequence data. University College Dublin (Ireland): Jim Carolan conducted proteomic analysis of aphid salivary glands. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The project has generated what will be part of the first published genome sequence of Myzus persicae (green peach aphid). Six salivary proteins that affect aphid reproduction on plants have been identified. Regulated expression of these proteins suggests that they function in aphid responses to particular host plant characteristics.
Publications
- No publications reported this period
|