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Molecular Studies of the Salivary Glands of the Pea Aphid, Acyrthosiphon Pisum (Harris)

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Molecular Studies of the Salivary Glands of the Pea Aphid, Acyrthosiphon Pisum (Harris) MOLECULAR STUDIES OF THE SALIVARY GLANDS OF THE PEA APHID, ACYRTHOSIPHON PISUM (HARRIS) by NAVDEEP S. MUTTI M. S., Punjab Agricultural University, India, 1998 AN ABSTRACT OF A DISSERTATION submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Entomology College of Agriculture KANSAS STATE UNIVERSITY Manhattan, Kansas 2006 ABSTRACT Salivary secretions are a key component of aphid-plant interactions. Aphids’ salivary proteins interact with plant tissues, gaining access to phloem sap and eliciting responses which may benefit the insect. In an effort to isolate and identify key components in salivary secretions, we created a salivary gland cDNA library. Several thousand randomly selected cDNA clones were sequenced. We grouped these sequences into 1769 sets of essentially identical sequences, or clusters. About 22% of the clusters matched clearly to (non-aphid) proteins of known function. Among our cDNAs, we have identified putative oxido-reductases and hydrolases that may be involved in the insect's attack on plant tissue. C002 represents an abundant transcript among the genes expressed in the salivary glands. This cDNA encodes a novel protein that fails to match to proteins outside of aphids and is of unknown function. In situ hybridization and immunohistochemistry localized C002 in the same sub-set of cells within the principal salivary gland. C002 protein was detected in fava beans that were exposed to aphids, verifying that C002 protein is a secreted protein. Injection of siC002-RNA caused depletion of C002 transcript levels dramatically over a 3 day period after injection. With a lag of 1 – 2 days, the siC002-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections of adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands. MOLECULAR STUDIES OF THE SALIVARY GLANDS OF THE PEA APHID, ACYRTHOSIPHON PISUM (HARRIS) by NAVDEEP S. MUTTI M. S., Punjab Agricultural University, India, 1998 A DISSERTATION submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Entomology College of Agriculture KANSAS STATE UNIVERSITY Manhattan, Kansas 2006 Approved by: Approved by: Co-Major Professor Co-Major Professor Dr. John C. Reese Dr. Gerald R. Reeck COPYRIGHT Navdeep S. Mutti 2006 ABSTRACT Salivary secretions are a key component of aphid-plant interactions. Aphids’ salivary proteins interact with plant tissues, gaining access to phloem sap and eliciting responses which may benefit the insect. In an effort to isolate and identify key components in salivary secretions, we created a salivary gland cDNA library. Several thousand randomly selected cDNA clones were sequenced. We grouped these sequences into 1769 sets of essentially identical sequences, or clusters. About 22% of the clusters matched clearly to (non-aphid) proteins of known function. Among our cDNAs, we have identified putative oxido-reductases and hydrolases that may be involved in the insect's attack on plant tissue. C002 represents an abundant transcript among the genes expressed in the salivary glands. This cDNA encodes a novel protein that fails to match to proteins outside of aphids and is of unknown function. In situ hybridization and immunohistochemistry localized C002 in the same sub-set of cells within the principal salivary gland. C002 protein was detected in fava beans that were exposed to aphids, verifying that C002 protein is a secreted protein. Injection of siC002-RNA caused depletion of C002 transcript levels dramatically over a 3 day period after injection. With a lag of 1 – 2 days, the siC002-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections of adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands. TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES.............................................................................................................. x ACKNOWLEDGEMENTS............................................................................................... xi DEDICATION.................................................................................................................. xii CHAPTER 1 .......................................................................................................................1 Chapter 1: Review of Literature ......................................................................................... 2 Aphid Taxonomy ............................................................................................................ 2 Aphid Feeding................................................................................................................. 3 Salivary Glands............................................................................................................... 6 Aphid Saliva and Salivary Proteins ................................................................................ 9 Plant Resistance and Defense Response to Aphids ...................................................... 14 Economic Importance of Aphids .................................................................................. 17 Aphids as Carriers of Viruses ....................................................................................... 18 Symbionts of Aphids..................................................................................................... 19 RNA Interference in Insects.......................................................................................... 22 Specific Objectives ........................................................................................................... 25 References..................................................................................................................... 26 CHAPTER 2 ..................................................................................................................... 58 Chapter 2: An EST library from Salivary Glands of the Pea Aphid, Acyrthosiphon pisum. ........................................................................................................................................... 59 Abstract......................................................................................................................... 60 Introduction................................................................................................................... 61 Material and Methods ................................................................................................... 62 Plants and Aphids ..................................................................................................... 62 Phagmid cDNA Library Construction ...................................................................... 62 Sequencing, Sequence Processing, and Annotation ................................................. 63 Results........................................................................................................................... 64 Salivary Transcript Catalog for A. pisum.................................................................. 64 vi Classification of Salivary Transcripts of A. pisum.................................................... 64 Proteases and Other Hydrolases in the Salivary Glands of A. pisum........................ 66 Oxidoreducatases in the Salivary Glands of A. pisum .............................................. 67 Discussion..................................................................................................................... 68 Acknowledgements....................................................................................................... 72 References..................................................................................................................... 73 Chapter 3: A Novel Transcript and Protein from the Salivary Glands of Pea Aphid, Acyrthosiphon pisum......................................................................................................... 96 Abstract......................................................................................................................... 97 Introduction................................................................................................................... 98 Material and Methods ................................................................................................... 99 Plants and Aphids ..................................................................................................... 99 Phagmid cDNA Library Construction and Sequencing.......................................... 100 RNA Isolation and RT-PCR ................................................................................... 100 RNA Isolation and Northern Blotting..................................................................... 101 Genomic DNA Isolation and Southern Blotting..................................................... 102 In situ Hybridization ............................................................................................... 102 Expression of Recombinant Protein in E. coli for Antibody Preparation............... 104 Western Blotting ....................................................................................................
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