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Developing Resources for Identification of Virus-Resistant Wheat Germplasm
Principal Investigators: Anna Whitfield, Department of Plant Pathology, Kansas State University, 785-532-3364, aewtospo@ksu.edu
Dallas Seifers, KSU Agricultural Research Center-Hays, 785- 625-3425
ext. 217, dseifers@ksu.edu
James Stack, Department of Plant Pathology, Kansas State University, 785-532-1388, jstack@ksu.edu
INTRODUCTION
Viruses are some of the most economically-important pathogens infecting wheat in Kansas. For example, just this past year, Kansas growers suffered an estimated $109 million in losses due to wheat streak mosaic virus alone (1). To date, genetic resistance is the most reliable and effective means for combating plant viruses and the search for new sources of virus-resistant wheat germplasm remains a primary focus at KSU. This important task requires the collaborative efforts of virologists, wheat geneticists, and plant breeders equipped with the state-of-the-art research tools for screening germplasm collections. Our long-range goal is to provide Kansas wheat scientists with the best virological research tools necessary for conducting resistance screening trials in the field and greenhouse. To this end, the specific aims of our research are to 1) establish the Kansas Wheat Virus Collection; 2) develop diagnostic molecular tools for identification of newly-emergent strains of High Plains virus (HPV); and 3) develop a vascular puncture inoculation (VPI) technique for identification of virus-resistant germplasm. Collectively, the products of our research will provide technologies and innovations that support the profitability of Kansas wheat producers and will improve the efficiencies of disease-resistance breeding programs at KSU.
RATIONALE AND SIGNIFICANCE
The goal of our proposed research is to find novel ways to overcome some of the technical hurdles faced by scientists searching for virus-resistant germplasm. We have identified some ways that will improve the tools we use to screen for resistance:
· As virologists we know that plant viruses are obligate parasites; they require a living host to survive. Because of this requirement, breeders depend on a constant supply of living virus-infected plants for their screening process. Maintaining active plant stocks is a labor-intensive and expensive task requiring the use of greenhouse space and intensive management of plant stocks. In some cases, important isolates can be lost due to rapid decline of virus-infected stocks. We propose to overcome this technical obstacle by maintaining a collection of live wheat viruses without depending on living plant tissues and growing conditions. This collection will be readily available to wheat breeders upon demand.
· Another obstacle virologists and breeders face is the lack of tools for identifying and monitoring newly emerging strains of wheat viruses. One such virus that poses a potential threat to Kansas wheat growers is High Plains virus (HPV). Since HPV was first discovered in Kansas in 1993, this virus has become a persistent disease problem for wheat producers. More recently, new isolates of HPV have emerged in Kansas; however we lack reliable diagnostic tools for universal detection of these new strains. Without proper detection tools for this virus, we are possibly underestimating their contribution to annual yield losses in growers' fields. Developing diagnostic tools will enable us to assess the impact of HPV on wheat production, design and implement IPM strategies, and identify HPV-resistant germplasm. In addition, we know that the greatest losses occur in wheat co-infected with both HPV and WSMV (1), and diagnostic tools will enable us to study the interactions between these viruses to better manage the HPV-WSMV-wheat curl mite disease complex.
· The process of screening germplasm for virus resistance requires efficient plant-inoculation techniques. Many viruses, like HPV, require arthropod vectors (such as the wheat curl mite) to be transmitted to their plant host, making it impossible for the experimenter to mechanically inoculate wheat leaf tissues with these recalcitrant viruses. We propose to develop a vascular puncture inoculation (VPI) technique for wheat to overcome this technical problem. The VPI is a cost-effective and high throughput method for screening resistance to viruses that cannot be mechanically inoculated by traditional methods. It is a well-established technique for maintaining viruses in corn and is being used for identification of disease resistant corn germplasm by USDA-Ohio State geneticists and breeders. To be successful in our screening projects, we need a comparable tool for wheat to be implemented by Kansas breeders.
PROCEDURES/METHODOLOGY
Our procedures and methodology are outlined in the following three objectives:
Objective 1. Establish the Kansas Wheat Virus Collection
Objective 2. Sequence HPV and develop diagnostic PCR primers.
Objective 3. Development of VPI for identification of virus-resistant germplasm.
Progress Report
Quarter 2 FY2008
Our long-range goal is to provide KS wheat scientists with the best virological research tools necessary for conducting resistance screening trials in the field and greenhouse. To this end, the specific aims of our research are to 1) establish the Kansas Wheat Virus Collection; 2) develop diagnostic molecular tools for identification of newly-emergent strains of High Plains virus (HPV); and 3) develop a vascular puncture inoculation (VPI) technique for identification of virus-resistant germplasm.
Since receiving our funding from the Wheat Commission Board in July 2007, we have made progress on each of our proposed research objectives in the following ways:
Aim 1 (establish the Kansas Wheat Virus Collection): We are continuing to store in our liquid nitrogen storage tank and -80 freezer KS virus isolates and antisera to HPV and BYDV for virus detection purposes. We acquired pure cultures of three different HPV isolates (HPV-06A, U04-82, and U04-83) in corn leaf tissue from Dallas Seifers (KSU Agricultural Research Center, Hays KS). Recently, we obtained another HPV isolate (U04-143) originally isolated by Dallas Seifers. We got this isolate into culture by vascular puncture inoculation (VPI) of sweet corn seeds (cv. Spirit) and it tested positive in serological tests (ELISA) using antisera raised against the original HPV strain (HPV-06A). With the help of the Plant Disease Diagnostic Clinic, our virus collection also includes symptomatic wheat tissue that tested positive for HPV in serological tests (ELISA). Our ultimate goal is to make these live viruses and virus detection tools readily available to scientists working towards screening wheat germplasm for resistance to viruses of economic importance to KS growers.
Aim 2 (develop diagnostic molecular tools for identification of newly-emergent strains of High Plains virus (HPV): In order to improve our ability to detect diverse isolates of HPV, we proposed to design “universal” PCR primers based on the genome sequences of three different HPV isolates that vary widely in their nucleocapsid protein sequence and differ significantly in their serological reaction with the original antibodies. To date, we have isolated double-stranded RNA (dsRNA) from corn tissue infected with U04-143 (reacts strongly with the antibody generated against the original isolate) and U04-83 (weak to no reaction with the antibody). dsRNA is the replicative intermediate form of RNA viruses (like HPV) and can be used as a platform for cloning of viral genes. We found that both isolates contain three to four dsRNA segments, ranging from 1200 – 2500 kilobases in length. We continue to work on our shotgun cloning of the genomes of these two different HPV isolates using the dsRNA platform. Ultimately, we will use this sequence information to generate PCR primers for accurate detection of HPV.
To complement our shotgun cloning project and to aid in goal to find molecular strategies for HPV detection, we have most recently tested PCR primers specific to the HPV-N gene (nucleocapsid gene sequence) and the HPV-L gene (polymerase gene). The HPV-N primers were designed and published by Lebas et al. (2004, Plant Disease, Volume 89:1103-1108). These primers are specific to the RNA3 gene sequence of HPV isolates that tested positive for the virus in serological tests (much like HPV-06A and U04-143). For the HPV-L gene primers, we designed PCR primers based on published sequence information for the RNA1 sequence of HPV obtained from a corn field isolate of the virus (Skare et al., 2006, Virology, Volume 347:343-353). To test the two primer pairs (N and L primers), we first isolated RNA from frozen and fresh sweet corn tissue infected with pure isolates of U0-082, U0-083, and U0-143. We performed reverse transcriptase (RT)-PCR on the RNA samples using a two step method: single-strand synthesis of cDNA with VERSO™ (ABGene) and then PCR amplification using the L and N-specific primer pairs. For the N-specific primers, we obtained the expected PCR product (approximately 339 bp size) for U0-143 only. For the L-specific primers, we obtained the expected PCR product (approximately 295 bp size) for U0-082 only (there are currently no serological tests available for detection of this HPV strain). Interestingly, there are no reports of using a PCR-based tool for detection HPV isolates that do not react to antisera raised against the original HPV isolate. Our results may lead to the development of a multiplex PCR technique for simultaneous detection of two HPV strains that are genotypically diverse.
Aim 3 (develop a vascular puncture inoculation (VPI) technique for identification of virus-resistant germplasm): Many viruses, like HPV, require arthropod vectors (such as the wheat curl mite) to be transmitted to their plant host, making it impossible for the experimenter to mechanically inoculate wheat leaf tissues with these recalcitrant viruses. HPV is not mechanically transmissible by leaf-rub-inoculation making it difficult to screen germplasm for virus resistance. Scientists have been able to transmit HPV by vascular puncture inoculation (VPI) to corn. In our hands, we have successfully used VPI to inoculate sweet corn (Spirit) seeds with HPV. We are currently working on optimizing the technique to increase our greenhouse stocks of HPV-infected plants for experimental purposes (to increase plant biomass for virus purification and to get 2008 field isolates into culture). We are also planning to collaborate with Mike Smith (Department of Entomology, K-State) to use the wheat curl mite to efficiently transmit HPV to wheat and sweet corn to bulk-up our virus stocks. Our ultimate goal is to use VPI and mite-transmission to facilitate the high-throughput screening of wheat germplasm for resistance to HPV and to investigate the interaction between HPV and wheat streak mosaic virus (WSMV) (Wheat Commission Grant FY08 submitted).
Dallas Seifers, KSU Agricultural Research Center-Hays, 785- 625-3425
ext. 217, dseifers@ksu.edu
James Stack, Department of Plant Pathology, Kansas State University, 785-532-1388, jstack@ksu.edu
INTRODUCTION
Viruses are some of the most economically-important pathogens infecting wheat in Kansas. For example, just this past year, Kansas growers suffered an estimated $109 million in losses due to wheat streak mosaic virus alone (1). To date, genetic resistance is the most reliable and effective means for combating plant viruses and the search for new sources of virus-resistant wheat germplasm remains a primary focus at KSU. This important task requires the collaborative efforts of virologists, wheat geneticists, and plant breeders equipped with the state-of-the-art research tools for screening germplasm collections. Our long-range goal is to provide Kansas wheat scientists with the best virological research tools necessary for conducting resistance screening trials in the field and greenhouse. To this end, the specific aims of our research are to 1) establish the Kansas Wheat Virus Collection; 2) develop diagnostic molecular tools for identification of newly-emergent strains of High Plains virus (HPV); and 3) develop a vascular puncture inoculation (VPI) technique for identification of virus-resistant germplasm. Collectively, the products of our research will provide technologies and innovations that support the profitability of Kansas wheat producers and will improve the efficiencies of disease-resistance breeding programs at KSU.
RATIONALE AND SIGNIFICANCE
The goal of our proposed research is to find novel ways to overcome some of the technical hurdles faced by scientists searching for virus-resistant germplasm. We have identified some ways that will improve the tools we use to screen for resistance:
· As virologists we know that plant viruses are obligate parasites; they require a living host to survive. Because of this requirement, breeders depend on a constant supply of living virus-infected plants for their screening process. Maintaining active plant stocks is a labor-intensive and expensive task requiring the use of greenhouse space and intensive management of plant stocks. In some cases, important isolates can be lost due to rapid decline of virus-infected stocks. We propose to overcome this technical obstacle by maintaining a collection of live wheat viruses without depending on living plant tissues and growing conditions. This collection will be readily available to wheat breeders upon demand.
· Another obstacle virologists and breeders face is the lack of tools for identifying and monitoring newly emerging strains of wheat viruses. One such virus that poses a potential threat to Kansas wheat growers is High Plains virus (HPV). Since HPV was first discovered in Kansas in 1993, this virus has become a persistent disease problem for wheat producers. More recently, new isolates of HPV have emerged in Kansas; however we lack reliable diagnostic tools for universal detection of these new strains. Without proper detection tools for this virus, we are possibly underestimating their contribution to annual yield losses in growers' fields. Developing diagnostic tools will enable us to assess the impact of HPV on wheat production, design and implement IPM strategies, and identify HPV-resistant germplasm. In addition, we know that the greatest losses occur in wheat co-infected with both HPV and WSMV (1), and diagnostic tools will enable us to study the interactions between these viruses to better manage the HPV-WSMV-wheat curl mite disease complex.
· The process of screening germplasm for virus resistance requires efficient plant-inoculation techniques. Many viruses, like HPV, require arthropod vectors (such as the wheat curl mite) to be transmitted to their plant host, making it impossible for the experimenter to mechanically inoculate wheat leaf tissues with these recalcitrant viruses. We propose to develop a vascular puncture inoculation (VPI) technique for wheat to overcome this technical problem. The VPI is a cost-effective and high throughput method for screening resistance to viruses that cannot be mechanically inoculated by traditional methods. It is a well-established technique for maintaining viruses in corn and is being used for identification of disease resistant corn germplasm by USDA-Ohio State geneticists and breeders. To be successful in our screening projects, we need a comparable tool for wheat to be implemented by Kansas breeders.
PROCEDURES/METHODOLOGY
Our procedures and methodology are outlined in the following three objectives:
Objective 1. Establish the Kansas Wheat Virus Collection
Objective 2. Sequence HPV and develop diagnostic PCR primers.
Objective 3. Development of VPI for identification of virus-resistant germplasm.
Progress Report
Quarter 2 FY2008
Our long-range goal is to provide KS wheat scientists with the best virological research tools necessary for conducting resistance screening trials in the field and greenhouse. To this end, the specific aims of our research are to 1) establish the Kansas Wheat Virus Collection; 2) develop diagnostic molecular tools for identification of newly-emergent strains of High Plains virus (HPV); and 3) develop a vascular puncture inoculation (VPI) technique for identification of virus-resistant germplasm.
Since receiving our funding from the Wheat Commission Board in July 2007, we have made progress on each of our proposed research objectives in the following ways:
Aim 1 (establish the Kansas Wheat Virus Collection): We are continuing to store in our liquid nitrogen storage tank and -80 freezer KS virus isolates and antisera to HPV and BYDV for virus detection purposes. We acquired pure cultures of three different HPV isolates (HPV-06A, U04-82, and U04-83) in corn leaf tissue from Dallas Seifers (KSU Agricultural Research Center, Hays KS). Recently, we obtained another HPV isolate (U04-143) originally isolated by Dallas Seifers. We got this isolate into culture by vascular puncture inoculation (VPI) of sweet corn seeds (cv. Spirit) and it tested positive in serological tests (ELISA) using antisera raised against the original HPV strain (HPV-06A). With the help of the Plant Disease Diagnostic Clinic, our virus collection also includes symptomatic wheat tissue that tested positive for HPV in serological tests (ELISA). Our ultimate goal is to make these live viruses and virus detection tools readily available to scientists working towards screening wheat germplasm for resistance to viruses of economic importance to KS growers.
Aim 2 (develop diagnostic molecular tools for identification of newly-emergent strains of High Plains virus (HPV): In order to improve our ability to detect diverse isolates of HPV, we proposed to design “universal” PCR primers based on the genome sequences of three different HPV isolates that vary widely in their nucleocapsid protein sequence and differ significantly in their serological reaction with the original antibodies. To date, we have isolated double-stranded RNA (dsRNA) from corn tissue infected with U04-143 (reacts strongly with the antibody generated against the original isolate) and U04-83 (weak to no reaction with the antibody). dsRNA is the replicative intermediate form of RNA viruses (like HPV) and can be used as a platform for cloning of viral genes. We found that both isolates contain three to four dsRNA segments, ranging from 1200 – 2500 kilobases in length. We continue to work on our shotgun cloning of the genomes of these two different HPV isolates using the dsRNA platform. Ultimately, we will use this sequence information to generate PCR primers for accurate detection of HPV.
To complement our shotgun cloning project and to aid in goal to find molecular strategies for HPV detection, we have most recently tested PCR primers specific to the HPV-N gene (nucleocapsid gene sequence) and the HPV-L gene (polymerase gene). The HPV-N primers were designed and published by Lebas et al. (2004, Plant Disease, Volume 89:1103-1108). These primers are specific to the RNA3 gene sequence of HPV isolates that tested positive for the virus in serological tests (much like HPV-06A and U04-143). For the HPV-L gene primers, we designed PCR primers based on published sequence information for the RNA1 sequence of HPV obtained from a corn field isolate of the virus (Skare et al., 2006, Virology, Volume 347:343-353). To test the two primer pairs (N and L primers), we first isolated RNA from frozen and fresh sweet corn tissue infected with pure isolates of U0-082, U0-083, and U0-143. We performed reverse transcriptase (RT)-PCR on the RNA samples using a two step method: single-strand synthesis of cDNA with VERSO™ (ABGene) and then PCR amplification using the L and N-specific primer pairs. For the N-specific primers, we obtained the expected PCR product (approximately 339 bp size) for U0-143 only. For the L-specific primers, we obtained the expected PCR product (approximately 295 bp size) for U0-082 only (there are currently no serological tests available for detection of this HPV strain). Interestingly, there are no reports of using a PCR-based tool for detection HPV isolates that do not react to antisera raised against the original HPV isolate. Our results may lead to the development of a multiplex PCR technique for simultaneous detection of two HPV strains that are genotypically diverse.
Aim 3 (develop a vascular puncture inoculation (VPI) technique for identification of virus-resistant germplasm): Many viruses, like HPV, require arthropod vectors (such as the wheat curl mite) to be transmitted to their plant host, making it impossible for the experimenter to mechanically inoculate wheat leaf tissues with these recalcitrant viruses. HPV is not mechanically transmissible by leaf-rub-inoculation making it difficult to screen germplasm for virus resistance. Scientists have been able to transmit HPV by vascular puncture inoculation (VPI) to corn. In our hands, we have successfully used VPI to inoculate sweet corn (Spirit) seeds with HPV. We are currently working on optimizing the technique to increase our greenhouse stocks of HPV-infected plants for experimental purposes (to increase plant biomass for virus purification and to get 2008 field isolates into culture). We are also planning to collaborate with Mike Smith (Department of Entomology, K-State) to use the wheat curl mite to efficiently transmit HPV to wheat and sweet corn to bulk-up our virus stocks. Our ultimate goal is to use VPI and mite-transmission to facilitate the high-throughput screening of wheat germplasm for resistance to HPV and to investigate the interaction between HPV and wheat streak mosaic virus (WSMV) (Wheat Commission Grant FY08 submitted).
