Progress 04/01/11 to 03/31/13
Outputs
Target Audience: The research community, both in academia and in the broader sense, particularly those involved in understanding plant-insect interactions and host-plant resistance, as better understanding of these areas has great potential for the development of rational pest management strategies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A MS student (Cherie Ognibene) was recruited and trained as part of this project and graduated in 2012. As well as receiving laboratory training she was able to attend and present at a number of national meetings to gain wider experience and build a professional network. A post-doctoral researcher (Sampurna Sattar) was recruited and gained significant experience and training in the analysis of next generation sequencing datasets as well as technical laboratory skills. Dr. Sattar gave several invited talks and was the primary author of two peer-reviewed articles. Three undergraduate assistants were employed on this grant; they were able to experience laboratory research first-hand and two of the students have enrolled in graduate school. How have the results been disseminated to communities of interest? In addition to the publication of several peer-reviewed articles, results from the project have been disseminated at national and international scientific meetings. The large data-sets generated have also been deposited in public data repositories where they are freely available to other researchers. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aphis gossypii resistance in Cucumis melo has been attributed to the presence of Vat, a single dominant gene belonging to the CC-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 regulating these gene responses. SmallRNA (sRNA) libraries were constructed from bulked leaf tissues of a Vat+ melon line following early and late aphid infestations. Sequence analysis indicated the expression profiles of conserved and newly identified miRNAs were altered during different stages of aphid herbivory. These results were verified by qPCR experiments in both resistant Vat+ and susceptible Vat- interactions. The comparative analyses revealed that most of the conserved miRNA families were differentially regulated during the early stages of aphid infestation in the resistant and susceptible interactions. Along with the conserved miRNA families, eighteen cucurbit-specific miRNAs were expressed during the different stages of aphid herbivory, of which five miRNAs were identified to be novel. The novel miRNAs were validated by qPCR and no significant difference in expression was observed between the early and late time points in the susceptible and resistant interactions. Putative targets of the conserved and novel miRNA were identified by computational analysis and melon degradome libraries were created to identify miRNA cleavage products. Initial analysis of a melon degradome library comprising 7,634,766 reads corresponded to 2,463,725 distinct sequences. Melon genes showing evidence of argonaute-catalyzed miRNA cleavage were detected using the CleaveLand pipeline version 3.0 for degradome sequence analysis. This analysis yielded a list of 409 miRNA:target pairs, including 84 unique miRNAs from 21 miRNA gene families and 86 distinct target transcripts corresponding to 70 unique genes. Along with the melon sRNA libraries, two insect sRNA libraries were generated from A. gossypii. sRNA libraries were constructed from A. gossypii collected from Vat+ and Vat- melon plants after 48 hours of feeding. Deep-sequencing data revealed the presence of 70 conserved, 12 aphid-specific, and nine novel miRNAs from both the libraries. The aphid Vat+ library showed a high abundance of piRNA sequences. Three of the novel miRNAs, validated by qPCR, were shown to be up-regulated and one down-regulated in the resistant interaction. Several plant miRNA families were also found in the aphid libraries. Predicted target genes for conserved and novel aphid miRNAs using in silico methods were categorized in into different functional groups. The majority of the target genes had functional roles in morphogenesis and anatomical structure determination. Genes belonging to the signal transduction pathway, cell differentiation and catabolic processes were frequently identified target gene categories. Objective 1: Small RNA libraries were constructed and analyzed in melon and A. gossypii. Large datasets were generated and analyzed and provided the basis for the subsequent miRNA analysis outlined in objectives 2 and 3 as well as a resource for further investigation of the role of other smallRNAs classes in resistance. In melon: 24 conserved miRNA families were found as well as 18 cucurbit specific miRNAs of which 5 were completely novel. In A. gossypii: eighty-one conserved miRNAs, twelve aphid-specific miRNAs, and nine novel miRNAs were identified. sRNA libraries made from A. gossypii tissues also revealed an unexpected abundance of 27 nt long sRNA sequences in the aphids feeding on Vat+ plants. These piRNA-like elements appear to be involved in the aphid response to resistance, but their exact function is still unclear. Putative target genes were identified using in silico approaches and in melon these were confirmed by sequencing a degradome library and 5’RACE. Objective 2: QRT-PCR was used to identify miRNAs differentially regulated during the resistant interaction in melon. A large number of miRNA families showed statistically significant differences in expression with nine families up-regulated during the resistant interaction, but differences in the expression of miR166 and miR2911 had the strongest statistical support. In A. gossypii library counts and QRT-PCR were used to identify differentially regulated miRNAs. miRNAs were shown to be differentially regulated, including four of the novel miRNAs discovered. The identification of targets for melon miRNAs has given additional insight into the resistant interaction between melon and A. gossypii. Differentially induced melon miRNAs target transcription factors and other genes controlling glutathione synthesis, auxin production/mediation as well as ethylene production, indicating how they affect resistance. In A. gossypii target predictions for identified miRNAs indicated changes were occurring in the expression of genes involved in anatomical morphogenesis and cell differentiation processes. Objective 3: Several MiRNA families of plant origin were detected in the aphid sRNA libraries (miR156/miR157, miR166, miR168, miR2911 and miR2916) suggesting they had been transferred to the aphid during feeding. Three of these miRNAs (miR156/miR157, miR166 and miR168) have previously been reported to be present in phloem sap. The presence of the remaining plant miRNAs (miR2911 and miR2916) in the phloem sap has not been confirmed; however, their identification in the aphid libraries suggests that these miRNAs are also present in the phloem sap of melon. Interestingly, only two plant miRNA families were detected from Vat+ aphids. This could reflect the well-documented observations that aphids on Vat+ plants spend less time ingesting phloem sap than aphids feeding on Vat- plants. Experiments utilizing artificial diet containing radio-labeled miRNA-mimics confirmed that sRNAs could be acquired by aphids while feeding and were stable during ingestion for up to 4 days. In vivo feeding assays using artificial diets containing siRNAs showed that aphid mortality and reproduction are decreased in a dose-dependent manner.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Sattar S, Addo-Quay C, Song Y, Anstead JA, Sunkar R, Thompson GA. (2012). Expression of small RNA in Aphis gossypii and its potential role in the resistance interaction with melon. PLOS ONE 7(11):e48579.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Sampurna S., Addo-Quaye C., Anstead J.A.,. and Thompson, G.A. (2013). Degradome analysis of miRNA-targeted transcripts in the Aphis gossypiimelon resistance interaction Electronic conference proceedings (abstract), Plant Biology 2013, Providence, Rhode Island.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Sampurna S., Addo-Quaye C., Song Y., Anstead J.A., Sunkar R. and Thompson, G.A. (2013). Small RNAs Involved In Vat-mediated resistance against Aphis gossypii in melon. Arthropod Genomics workshop, Plant and Animal Genome Conference, San Diego 2013.
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Progress 04/01/11 to 03/31/12
Outputs
OUTPUTS: Aphis gossypii resistance in Cucumis melo has been attributed to the presence of Vat, a single dominant gene belonging to the CC-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. SmallRNA (sRNA) libraries were constructed from bulked leaf tissues of a Vat+ melon line following early and late aphid infestations. Sequence analysis indicated the expression profiles of conserved and newly identified miRNAs were altered during different stages of aphid herbivory. These results were verified by qPCR experiments in both resistant Vat+ and susceptible Vat- interactions. The comparative analyses revealed that most of the conserved miRNA families were differentially regulated during the early stages of aphid infestation in the resistant and susceptible interactions. Along with the conserved miRNA families, eighteen cucurbit-specific miRNAs were expressed during the different stages of aphid herbivory, of which five miRNAs were identified to be novel. The novel miRNAs were validated by qPCR and no significant difference in expression was observed between the early and late time points in the susceptible and resistant interactions. Targets of the conserved and novel miRNA were identified by computational analysis and some of the targets were verified by 5'RACE. Along with the melon sRNA libraries, two libraries were also generated from A. gossypii. sRNA libraries were constructed from A. gossypii collected from Vat+ and Vat- melon plants after 48 hours of feeding. Deep-sequencing data revealed the presence of 70 conserved, 12 aphid-specific, and nine novel miRNAs from both the libraries. The aphid Vat+ library showed a high abundance of piRNA sequences. Three of the novel miRNAs, validated by qPCR, were shown to be up-regulated and one down-regulated in the resistant interaction. Several plant miRNA families were also found in the aphid libraries. Predicted target genes for conserved and novel aphid miRNAs using in-silico methods were categorized in into different functional groups. The majority of the target genes had functional roles in morphogenesis and anatomical structure determination. Genes belonging to the signal transduction pathway, cell differentiation and catabolic processes were frequently identified target gene categories. PARTICIPANTS: Dr. Gary A. Thompson, PI, Professor; Dr. Ramanjulu Sunkar, co-PI, Assistant Professor; Dr. James Anstead, Research Associate; Dr. Sampurna Sattar, Postdoctoral Fellow; Dr. Charles Addo-Quaye, Postdoctoral Fellow; and Ms. Cherie Ognibene, M.S. Student. Dr.'s Thompson, Anstead, Sattar and Addo-Quaye are members of the College of Agricultural Sciences at The Pennsylvania State University. All other participants are members of the Department of Biochemistry and Molecular Biology at Oklahoma State University. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Isolation and biochemical characterization of genes 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 to other important crops. As the name implies, the cotton-melon aphid is also a major pest of cotton, and the melon resistance or defense genes could serve as a course of aphid resistance for cotton. Chemical treatment is the primary method of controlling and managing this pest in cotton. However, the development of aphid resistant cotton in combination with biological controls, such as the very effecitve aphid fungus, would significantly lower the production and environmental costs associated with controlling outbreaks of this insect pest. The identification of smallRNA genes involved in aphid growth and devlopment will provide aditional targets for aphid control as well as deepen our understanding of aphid growth and migration with important consequences for outbreak prediction and viral transmission.
Publications
- Sattar, S., Y. Song, J. A. Anstead, R. Sunkar, and G. A. Thompson. 2012. Cucumis melo MicroRNA Expression Profile During Aphid Herbivory in a Resistant and Susceptible Interaction. Molecular Plant-Microbe Interactions 25: 839-848.
- Ognibene, C. 2012. Developing a Method to Screen Potential Targets for Artificial RNAi in Aphids to be Used as a New Type of Resistance in Plants. M.S. Thesis. Oklahoma State University. Stillwater, OK. 107 pp.
- Sattar, S., Y. Song, J. Anstead, R. Sunkar, and G. Thompson. 2012. MicroRNA profiling in melon during aphid herbivory in susceptible and resistant interactions. Electronic conference proceedings. Plant Interaction with Pest and Pathogen Workshop, Plant and Animal Genome Conference 2012. San Diego, CA. (Abstract PW536).
- Song, N., J. Liu, G. Chakraborty, and J. Anstead. 2012. Investigating Host Plant Resistance to Aphid Feeding through SAS Text Miner. Proceedings of the SAS Global Forum 2012 Conference, Orlando, Florida. P 209-2012.
- Sattar, S., R. Sunkar, and G. Thompson. 2011. Differential Expression of miRNAs during early and late stages of aphid infestation on a resistant host. Electronic conference proceedings. Plant and Animal Genome 2011. San Diego, CA. (Abstract P760).
- Sattar, S., R. Sunkar, J. Anstead, and G. Thompson. 2011. Expression profile of miRNA during aphid herbivory. Electronic conference proceedings. Plant Biology 2011. Minneapolis MN. (Abstract P19048).
- Anstead, J., S. Sattar, R. Sunkar, and G. Thompson. 2011. Small RNAs and their targets are differentially expressed in susceptible and resistant interaction between Aphis gossypii and Cucumis melo. Electronic conference proceedings. Annual Meeting of Entomological Society of America. Reno, NV. (Abstract D0306).
- Sattar, S., J. Anstead, R. Sunkar, and G. Thompson. 2012. MicroRNA, transcription factor, and defense gene expression during susceptible and resistant aphid interactions in melon. Electronic conference proceedings. Plant and Animal Genome Conference 2012. San Diego, CA. (Abstract PO828).
- Sattar, S., J. Anstead, R. Sunkar, and G. Thompson. 2012. miRNA mediated regulation during aphid herbivory in susceptible and resistant interactions in melon. Electronic conference proceedings. Plant Biology 2012. Austin, TX. (Abstract P25046).
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