Progress 01/15/09 to 03/31/11
Outputs
OUTPUTS: Aphis gossypii resistance in Cucumis melo has been attributed to the presence of Vat, a single dominant gene belonging to the NBS-LRR family of resistance genes. Previous data showed that significant transcriptional reprogramming occurs in Vat+ plants during aphid infestations. Based on these observations, it was hypothesized that miRNAs are involved in fine tuning these gene responses. Small RNA libraries were constructed from bulked leaf tissues of a Vat+ melon line following early (2, 4 and 6 h) and late (8, 10, and 12 h) aphid infestations. A non-infested control library was used to study the basal level of miRNA expression in the Vat+ melon line. Illumina sequencing of the libraries revealed conserved miRNAs that were differentially expressed in the early and late time periods of aphid infestations, different microRNA's showed different patterns of response to aphid feeding, including transient up-regulation, sustained regulation and down-regulation. Northern blots and quantitative real-time PCR of the conserved miRNAs confirmed most of the trends observed within the library data. These data indicate that miRNAs influence the expression levels of a significant number of genes during the early stages of aphid herbivory. Some of the melon genes targeted by these miRNA were confirmed by RACE, and others will be confirmed from target libraries which are presently being constructed. A large number of putative novel miRNA's were also detected in the small RNA libraries, in silico analysis is being used to determine the validity of these sequences which will then be experimentally confirmed. Previous experiments had also shown that labeled smRNAs ingested by A. gossypii from artificial diets accumulated in the insect and were excreted in the honeydew. Two insect smRNA libraries were constructed from A. gossypii low molecular weight RNA to determine whether plant smRNAs in the phloem sap of melons are ingested by the insect, potentially impacting aphid gene expression. Aphids were allowed to feed for 48 h on either susceptible (vat-) or resistant (Vat+) melon breeding lines and collected for RNA extraction. Initial analysis of Illumina deep sequencing data obtained from the two libraries revealed the presence of melon miRNAs along with miRNAs previously identified in pea aphids and other insects. The melon miRNAs identified were among the most highly expressed in melon leaf tissue, and several of them were highly responsive to aphid feeding. These data have and continue to be disseminated to the wider scientific audience at national meetings and several manuscripts are in preperation. PARTICIPANTS: Dr. Gary A. Thompson, PI, Professor and Head Dr. Ramanjulu Sunkar, co-PI, Assistant Professor Dr. James Anstead, Senior Research Specialist Dr. Sampurna Sattar, Postdoctoral Fellow Dr. Guru Jagadeeswaran, Postdoctoral Fellow Ms. Cherie Ognibene, Ph.D. Student. All participants are members of the Department of Biochemistry and Molecular Biology at Oklahoma State University TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Identification of melon miRNAs will provide additional tools for plant breeders as well as expanding our understanding of the regulation of gene expression in this system. The elucidation of how gene expression responds to phloem feeding herbivores gives us a better understanding of how resistance functions in this model system and also opens the door for the manipulation of these responses to enhance host plant resistance. The identification of plant microRNAs transferred from plant to aphid also indicates it might be possible to use naturally expressed RNAs as plant defense molecules.
Publications
- Sattar S, Sunkar R, Thompson G A. Differential expression of miRNAs in early and late stages of aphid infestation in a resistant host Plant and Animal Genome XVIII Conference 2011. Abstract and Poster.
- Ognibene C, Sattar S, Sunkar R and Thompson G A. Determining the role of smallRNA in phloem localized aphid resistance. International Conference on Plant Vascular Biology 2010. Abstract and Poster.
- Sattar S, Anstead J, Jagadeeswaran G, Sunkar R and Thompson G A. MicroRNA regulation of host plant resistance to aphids. Plant and Animal Genome XVIII Conference 2010. Abstract and Poster.
- Sattar S, Anstead J, Ognibene C and Thompson G A . Differences in smRNA expression profiles in Aphis gossypii in susceptible and resistant interactions with Cucumis melo. Annual meeting of Entomological Society of America 2010. Abstract and Poster.
- Anstead J, Song N, Thompson GA. Plant defense responses to Aphis gossypii (cotton-melon aphid) feeding in resistant and susceptible melon (Cucumis melo). Annual meeting of Entomological Society of America 2009. Abstract and Poster
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Progress 01/15/10 to 01/14/11
Outputs
OUTPUTS: Aphis gossypii resistance in Cucumis melo has been attributed to the presence of Vat, a single dominant gene belonging to the NBS-LRR family of resistance genes. Previous data showed that significant transcriptional reprogramming occurs in Vat+ plants during aphid infestations. Based on these observations, it was hypothesized that miRNAs are involved in fine tuning these gene responses. Small RNA libraries were constructed from bulked leaf tissues of a Vat+ melon line following early (2, 4 and 6 h) and late (8, 10, and 12 h) aphid infestations. A non-infested control library was used to study the basal level of miRNA expression in the Vat+ melon line. Illumina sequencing of the libraries revealed conserved miRNAs that were differentially expressed in the early and late time periods of aphid infestations. Expression of most of the conserved miRNAs peaked at the early stages of aphid infestation, but returned near the basal levels of expression during the later stages. Northern blots of the conserved miRNAs confirmed the trends observed with the library data. These data indicate that miRNAs influence the expression levels of a significant number of genes during the early stages of aphid herbivory. The melon genes targeted by these miRNA were confirmed by RACE, and their expression patterns evaluated by qRT-PCR experiments. Previous experiments had also shown that labeled smRNAs ingested by A. gossypii from artificial diets accumulated in the insect and were excreted in the honeydew. Two insect smRNA libraries were constructed from A. gossypii low molecular weight RNA to determine whether plant smRNAs in the phloem sap of melons are ingested by the insect, potentially impacting aphid gene expression. Aphids were allowed to feed for 48 h on either susceptible (vat-) or resistant (Vat+) melon breeding lines and collected for RNA extraction. Initial analysis of Illumina deep sequencing data obtained from the two libraries revealed the presence of melon miRNAs along with miRNAs previously identified in pea aphids and other insects. PARTICIPANTS: Dr. Gary A. Thompson, PI, Professor and Head; Dr. Ramanjulu Sunkar, co-PI, Assistant Professor; Dr. James Anstead, Senior Research Specialist; Dr. Sampurna Sattar, Postdoctoral Fellow; Dr. Guru Jagadeeswaran, Postdoctoral Fellow; Ms. Cherie Ognibene, Ph.D. Student. All participants are members of the Department of Biochemistry and Molecular Biology at Oklahoma State University TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Identification of the molecular mechanisms involved in aphid resistance will provide the basis for future work to determine the physiological mechanism of aphid resistance and to transfer defense or resistance genes in to other crops. Chemical treatment is the primary method of controlling and managing this pest, however, the development of aphid resistant crops in combination with biological controls, such as the very effect aphid fungus, would significantly lower the production and environmental costs associated with controlling outbreaks of this insect pest.
Publications
- Sattar S, Anstead J, Jagadeeswaran G, Sunkar R and Thompson G A. MicroRNA regulation of host plant resistance to aphids. Plant and Animal Genome XVIII Conference 2010. Abstract and Poster.
- Ognibene C, Sattar S, Sunkar R and Thompson G A. Determining the role of smallRNA in phloem localized aphid resistance. International Conference on Plant Vascular Biology 2010. Abstract and Poster.
- Sattar S, Anstead J, Ognibene C and Thompson G A . Differences in smRNA expression profiles in Aphis gossypii in susceptible and resistant interactions with Cucumis melo. Annual meeting of Entomological Society of America 2010. Abstract and Poster.
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Progress 01/15/09 to 01/14/10
Outputs
OUTPUTS: Resistance to Aphis gossypii (cotton-melon aphid) in Cucumis melo, is attributed to the single dominant gene Vat (virus aphid transmission). Vat is a member of the NBS-LRR family of genes conferring resistance to A. gossypii and non-persistent viruses transmitted by these aphids. Studies on differential gene expression in melon plants in response to aphid infestation have shown activation of several defense pathways. Emerging data indicates that small RNAs, especially microRNAs (miRNA), have diverse and important roles in regulating gene expression by translational repression in both plants and animals, including responses to insect herbivory. This project is designed to test the hypothesis that specific small RNAs regulate gene expression under the biotic stress caused by aphid feeding and are components of the resistance mechanism both as gene expression regulators and as plant defense molecules. The specific objectives are to: 1) construct small RNA libraries to identify small regulatory RNAs and their candidate targets in melon and A. gossypii; 2) characterize small RNAs that are responsive to aphid feeding in melon and determine if they are involved in A. gossypii resistance, and 3) determine if plant small RNAs are transferred during A. gossypii feeding and their effect on target genes in the aphid. To fulfill the first objective and to investigate the role of specific miRNAs and their target genes in aphid resistance, miRNA expression profiles are being analyzed in the aphid resistant melon breeding line AR5. To have a better understanding of the early and late regulated genes in the resistance mechanism, multiple miRNA libraries were constructed from melon leaf tissue collected at different time points (early: 2h,4h,6h & late: 8h,10h,12h) post aphid infestation and a no infestation control. Two other libraries were constructed from aphid tissues, which were feeding for 48 hours on either nearly isogenic resistant or susceptible melon plants. High throughput Illumina deep sequencing data from melon leaf small RNA libraries has been obtained, and the bioinformatic analysis of deep sequencing data is underway. Initial experiments were conducted to establish whether ingestion is an effective mode of acquiring small RNAs that subsequently influence aphid gene expression. Using the RNAi approach, 24 mer siRNA was designed against aphid gut-specific cathepsin and administered to the insect via artificial diet. The insects were collected after 24 and 48 h post feeding and the level of the cathepsin transcripts were monitored by qRT-PCR. The result of this experiment is currently being repeated for further statistical analysis. Additional aphid genes are being targeted for RNAi to establish this to be a valid mode of transfer of small RNA into the aphid gut. PARTICIPANTS: Dr. Gary A. Thompson, PI, Professor and Head; Dr. Ramanjulu Sunkar, co-PI, Assistant Professor; Dr. James Anstead, Senior Research Specialist; Dr. Sampurna Sattar, Postdoctoral Fellow; Dr. Guru Jagadeeswaran, Postdoctoral Fellow; Ms. Cherie Ognibene, Ph.D. Student. All participants are members of the Department of Biochemistry and Molecular Biology at Oklahoma State University TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Identification of the molecular mechanisms involved in aphid resistance will provide the basis for future work to determine the physiological mechanism of aphid resistance and to transfer defense or resistance genes in to other crops. Chemical treatment is the primary method of controlling and managing this pest, however, the development of aphid resistant crops in combination with biological controls, such as the very effect aphid fungus, would significantly lower the production and environmental costs associated with controlling outbreaks of this insect pest.
Publications
- Anstead J., Samuel P., Song N., Wu C., Thompson G.A., and Goggin, F. 2010. Ethylene pathway activation in response to aphid feeding on resistant and susceptible melon and tomato plants. Entomol. Exp. Appl. 134: 170-181.
- Anstead J, Song N, Thompson GA. 2009. Plant defense responses to Aphis gossypii (cotton-melon aphid) feeding in resistant and susceptible melon (Cucumis melo). 57th Annual Meeting,
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