Small Grains Viral Disease Laboratory

The intent of these studies is to increase our understanding of the genetic and molecular basis of virus-host plant interactions in small grain cereal crops. This research emphasizes barley yellow dwarf virus (BYDV) because it is the most endemic and severe viral disease in small grain crops. Initial studies will focus on the use of BYDV viral genes as possible resistance genes, molecular genetic aspects of BYDV disease resistance genes found in wheat and barley, and the effect of viral disease on plant metabolism. The long term objective is to use this knowledge to develop virus resistant germplasm and cultivars as a means of reducing losses in grain quality and yield due to virus infection. The Small Grain Virus-Host Plant Interaction Laboratory is located within the Crop Production and Pest Control Research Unit of the USDA-Agricultural Research Service, on the Purdue University campus in West Lafayette, Indiana. This research is a component of a CRIS project entitled Molecular and Genetic Mechanisms of Resistance to Barley Yellow Dwarf Virus, which was initiated in 1996.

Specific Objectives:

A) Obtain transgenic barley and wheat plants containing BYDV viral genes and determine if these viral genes will inhibit virus establishment or replication.

B) Molecular genetic characterization of BYDV resistance in wheat.

C) Analyze carbohydrate metabolism in healthy and infected plants to determine how virus infection specifically alters a basic aspect of plant growth.

Need for Research

Small grain cereal crops such as wheat, barley and oats are very important agricultural commodities in the United States and world-wide. Wheat is the major grain constituent in bread, flour, and pastry foods and barley alone represents approximately $129 billion dollars in total business activity in the USA. In the USA wheat, barley and oats were harvested in 1990-1991 from approximately 28, 3 and 2.4 million hectares, respectively, while world-wide the figures are 231, 73, and 22 million hectares. Barley yellow dwarf virus (BYDV) causes various symptoms such as leaf yellowing, reduced tillering, and reduced root growth, all of which lowers yield and seed quality. When environmental conditions favor aphid infestation, BYDV is a serious problem. It has been estimated that losses due to natural infection can be as high as 30 to 50%. Furthermore, even in those years when there is not a major outbreak of BYDV, there appears to be a base level of BYDV infection which results in an unperceived loss of 1-5%. In wheat, a 1 to 5 % loss in yield, would decrease the level of production in the US by approximately 0.75 to 3.7 million tons, which in today's prices is a $123 to $615 million dollar loss. These figures illustrate that BYDV is a serious problem in the US and that it is one of the most serious viral pathogens of these small grain cereals world-wide.

At this point, there is little effective control due to the lack of comprehensive resistance genes in wheat, barley, and oat because of variation from year to year in the proportions of different BYDV strains. This makes it difficult to breed for resistance to all or even the most prevalent BYDV serotypes. In addition, very little is known about how BYDV infection alters the regulation of carbohydrate metabolism and ultimately photosynthesis. This is highly significant when one considers that carbohydrate metabolism has a central role in almost all aspects of plant growth/development and that starch is the predominant constituent in seeds of all cereal crops. This research, therefore, would fill a significant void in cereal crop research by elucidating BYDV resistance mechanisms already present in plants, utilizing novel methods to obtain BYDV resistance, and by enhancing our understanding of how BYDV infection reduces yield through an analysis of carbohydrate metabolism.

The knowledge of plant host-encoded and bioengineered viral resistance mechanisms and the technology to modify plant germplasm acquired from this research will allow us to direct and enhance crop productivity and quality. This research, therefore, has particular relevance to the mission of the ARS which is to "develop means to maintain and increase crop productivity". In addition to addressing the ARS mission as a whole, identifying, isolating, characterizing, and modifying genes involved in virus resistance and yield specifically meets the goals delineated as areas of emphasis in the ARS six-year program plan.

Research Approach

Understanding virus-host plant interactions and obtaining BYDV resistant germplasm will be approached in three different and complimentary directions: direct bioengineering of resistance, characterization of molecular markers associated with host-encoded resistance for eventual use in breeding schemes and bioengineering, and analysis of how virus infection affects carbohydrate metabolism. Although the effect of virus infection on plant metabolism is an important area, the primary emphasis of this research will focus on bioengineering of resistance and identification of molecular markers closely linked with host-encoded resistance.

Current Research Projects include:

I.Bioengineered Resistance

Initial efforts are focusing on the use of viral genes as a means of bioengineering resistant wheat and barley lines. We will be utilizing a replicase-mediated approach in which viral genes involved in replication, such as the polymerase genes, are used as the resistance genes. Previous work by myself and others have shown that replicase-mediated resistance gives complete or near immunity to the virus from which the replicase gene originated and related strains. This immunity is a much higher level of protection than that generally afforded by coat-protein-mediated protection. Furthermore, coat-protein-mediated protection has thus far not been successful for BYDV in barley and oats. The type of plant expression cassette that is being used consists of a dual 35S CaMV promoter-AdhI intron-viral gene-NOS terminator. The BYDV viral genes which will be inserted into this cassette will be the replicase genes of the group I strain PAV and/or the group II strain RPV as either intact genes or modified versions to determine which one is the most effective construct. Several of these have been made and utilized in wheat transformation experiments. Because BYDV is a phloem-limited virus other tissue-specific promoters such as the sucrose synthase, rice tungro bacilliform virus, and commelina yellow mottle virus promoters may be evaluated as potential promoters.

For the present, our focus is on wheat transformation. Currently we are transforming wheat through the use of the biolistic bombardment protocol. Because the efficiency of this procedure is quite low (1% success rate) we are also collaborating with Dr. Troy Weeks, USDA-ARS, Lincoln, NE to facilitate this process. Once transgenic plants are obtained, they will be analyzed for presence and expression of the viral gene, inoculated with BYDV via the appropriate aphid vector and the virus titer measured by ELISA two to three weeks after infection. This inoculation and testing system is in use at this time. Resistant lines will be further characterized to determine the level of resistance in greenhouse and field tests and correlate this with copy number, gene expression and mendelian genetics information.

II.Characterization of Host-encoded Resistance

At this point there is little effective control of BYDV in wheat, barley, and oats due to the lack of comprehensive resistance genes. Drs. Ohm and Sharma, here at Purdue University, are one of three groups in the world to have successfully introgressed a wheatgrass chromosome from Thinopyrum intermedium which provides BYDV resistance into elite wheat lines (Triticum aestivum). This project has three main goals:

1. To determine the level and spectrum of this resistance to the various strains of BYDV both in the original wheatgrass and in the wheat lines containing the wheatgrass chromosome.
2. Utilizing molecular markers previously mapped in wheat and other small grain crops make a map of the wheatgrass chromosome which gives resistance to BYDV. Because, the whole wheatgrass chromosome is not desirable for use in breeding for BYDV resistance analyze putative translocation lines to determine if they contain a reduced amount of the wheatgrass chromosome. Translocation lines which contain the least amount of the wheatgrass chromosome will be used for developing soft red winter wheat elite germplasm that is BYDV resistant. Molecular markers from this study will also be developed for use in marker- assisted selection of BYDV resistance.
3. Characterize the resistance response at the level of gene expression (absence or presence of mRNAs in resistant and susceptible lines) and determine if there are particular host proteins which interact with the virus in resistant lines and not susceptible lines. the presence or absence of protein profile. Determining the differences in resistant vs. susceptible lines at these levels will contribute to our understanding of how the virus interacts with its wheat host and extend our knowledge about the resistance mechanism itself. This characterization will utilize differential display analysis and a two-hybrid protein (interaction-trap) system. Differential display will display differences in the mRNA populations between susceptible and resistant lines through the use of a subtractive hybridization/polymerase chain reaction amplification strategy. The two-hybrid system is an in vivo method for screening for host-encoded proteins which interact with a bait protein such as the BYDV replicase, movement, or coat- protein.

III.Effect of Virus Infection on Carbohydrate Metabolism

It is well documented that BYDV is a phloem limited virus which reduces phloem flow by necrotic obliteration of the various cells associated with the phloem such as sieve elements, parenchyma, and companion cells. The subsequent restriction of the phloem leads to a number of significant changes such as reduced photosynthesis, increased respiration, and a reduction in translocation of carbohydrates from the leaves. One of the central questions in plant metabolism is the relationship between source and sink tissue because of its fundamental role in plant growth and yield. We are studying the effect of BYDV infection on the regulation of starch production in the barley seed. Starch is the major component in barley and all other small grain seeds and, therefore, is a major component of yield and quality. Our initial efforts have focused on determining the regulation and level of starch produced in seeds of healthy uninfected barley cultivars.

LAB PERSONNEL

LAB FACILITIES

Dr. Anderson has approximately 950 square feet of laboratory space fully equipped for molecular biology including the following major equipment: 96-well Perkin Elmer 9600 PCR thermocycler, gel electrophoresis apparatus, power supplies, sterile air hood, ultralow freezer, microcentrifuges, low speed and ultra-centrifuges with appropriate swinging bucket, fixed angle and near-vertical rotors, shaking incubators, stationary incubators, sequencing gel apparatus, cell electroporator, darkroom for gel documentation, UV-visible diode-array spectrophotometer with stand alone Hewlett-Packard computer, plant tissue culture incubators, fume hoods, water purification system, IBM compatible computer connected to the university mainframe computer for database connectivity, laser printer, DNA/protein analysis hardware/software, scintillation counter, speedvac, hybridization chambers and dedicated radioactive use room. Equipment within the Crop Production and Pest Control Research Unit and in the Agronomy department that is available to this project includes an automated X-ray film developer, oligonucleotide synthesizer, automated DNA sequencer, general photography darkroom, and additional incubators and sterile air hoods. Plant Growth Facilities: 1,000 sq. ft. of greenhouse space and approximately 88 sq. ft. of growth chamber space are available for rearing viruliferous aphids, transgenic plant studies, and growing plants as needed.

RECENT RESULTS

I.Bioengineered Resistance

Initial efforts have focused on the use of viral replicase genes as a means of bioengineering resistant wheat lines. Currently we are transforming wheat in the Small Grain Virus-Host Plant Interaction Laboratory through the use of the biolistic bombardment protocol. Because the efficiency of this procedure is relatively low (1% success rate) we are also collaborating with Dr. Troy Weeks, USDA-ARS, Lincoln, NE to facilitate this process. We are currently evaluating 15 transgenic lines for resistance and susceptibility to the PAV strain of BYDV as well as transgene expression at the RNA and protein levels. As more independent transformants are generated they are also evaluated for resistance. Those lines which are resistant will be further characterized to determine the level of resistance in greenhouse and field tests and correlate this with copy number, gene expression and mendelian genetics information.

II.Characterization of Host-encoded Resistance

1. We have determined that a single wheatgrass chromosome in wheat engenders complete resistance to subgroup II BYDV strains in all tissues. However, this same chromosome gives only partial resistance to subgroup I BYDV strains (PAV) in leaf and stem tissue and no protection in root tissue.
2. Utilizing molecular markers previously mapped in wheat and other small grain crops we have completed a structural analysis and have constructed a map of the
   wheatgrass chromosome which gives resistance to BYDV. See below the abstract of manuscript entitled "Structural organization of an alien Thinopyrum intermedium group 7 chromosome in US soft red winter wheat (Triticum aestivum L.)" which was submitted to Genome.
   

   Fluorescence in situ hybridization. Wheat chromosomes are red, while the bits of T. intermedium at the tip of one chromosome are green.

   
   However, the whole wheatgrass chromosome is not desirable for use in breeding for BYDV resistance. Consequently, in conjunction with constructing this molecular map, we have analyzed putative translocation lines to determine if they contain a reduced amount of the wheatgrass chromosome. Translocation lines which contain the least amount of the wheatgrass chromosome will be used for developing soft red winter wheat elite germplasm that is BYDV resistant. Molecular markers from this study will also be developed for use in marker-assisted selection of BYDV resistance. This work was recently presented at the Plant & Animal Genome V meeting. See abstract below.
3. To examine the differences in resistant vs. susceptible lines at these levels we are utilizing differential display analysis and a two-hybrid protein (interaction-trap) system. We are currently analyzing approximately 70 cDNA clones derived from the differential display analysis to determine if they are involved in some aspect of the resistance mechanism. The two-hybrid system is now functional and we are using various genes from the virus as baits to detect host-encoded proteins which interact with these viral proteins as a means of delineating this virus-plant interaction.

III.Effect of Virus Infection on Carbohydrate Metabolism

We are studying the effect of BYDV infection on the regulation of starch production in the barley seed because starch is the major constituent in barley and small grain seeds and, therefore, is a major yield and quality component. Our initial efforts have focused on determining the regulation and level of starch produced in seeds of healthy uninfected barley cultivars to get some basic information prior to examining infected plant lines. This work has been submitted to Plant Physiology.

Abstracts of recent research results are provided below:

M.G. Francki, O.R. Crasta, H.C Sharma, H.W Ohm, and J.M. Anderson. 1997. Structural organization of an alien Thinopyrum intermedium group 7 chromosome in US soft red winter wheat (Triticum aestivum L.). Submitted to Genome.

Abstract Barley Yellow Dwarf Virus (BYDV) resistance in soft red winter wheat (SRWW) cultivars has been achieved by substituting a group 7 chromosome from Thinopyrum intermedium for chromosome 7D. To localize BYDV resistance, a detailed molecular genetic analysis was done on the alien group 7 Th. intermedium chromosome to determine its structural organization. Triticeae group 7 RFLP markers and rye-specific repetitive sequences used in the analysis showed that the alien chromosome in the P29 substitution line has distinguishing features. The 350-480 bp rye telomeric sequence family was present on the long arm as determined by Southern and fluorescence in situ hybridization. However, further analysis using rye dispersed repetitive sequences indicated that this alien chromosome does not contain introgressed segments from the rye genome. The alien chromosome is homeologous to wheat 7A and 7D as determined by RFLP analysis. Presence of the waxy gene on 7A, 7B and 7D chromosomes but its absence on the alien chromosome in P29 suggest some internal structural differences on the short arm between Th. intermedium and wheat group 7 chromosomes. The identification of rye telomeric sequences on the alien Thinopyrum chromosome and the homeology to wheat chromosomes 7A and 7D provide the necessary information and tools to analyze smaller segments of the Thinopyrum chromosome and localize BYDV resistance in SRWW cultivars.

Anderson, J.M., O. Crasta, M. Francki, D. Bucholtz, H. Sharma and H.W. Ohm. 1997. Molecular and cytogenetic analysis of barley yellow dwarf virus resistant translocation lines containing Thinopyrum intermedium chromosomal segments. Plant and Animal Genome V. Jan. 12-17, San Diego, CA.

Abstract Wheat germplasm lacks true resistance to barley yellow dwarf virus (BYDV), the most significant viral pathogen of cereals worldwide. Soft red winter wheat containing the Thinopyrum intermedium 7E chromosome have been developed and determined to be resistant to BYDV. Translocation lines (TLs) have been developed by subjecting the monosomic alien substitution line to g-irradiation and monitoring the selfed progenies for chromosome number, segregation pattern of BYDV resistance and presence of Thinopyrum chromatin. The objectives of this research are qualitative and quantitative estimation of the presence of Thinopyrum chromatin in TLs, identification of the chromosomal segment containing BYDV resistance and identification of suitable translocation lines as germplasm for variety development and for use in targeted mapping strategies to isolate DNA markers associated with the BYDV resistance locus ( BYDVR). We have utilized a comparative mapping strategy to characterize the TLs. DNA markers shown to be collinear across several species of Gramineae or mapped on wheat group 7 chromosomes were utilized in identifying 7E and 7D chromosomal segments in TLs. The presence or absence of the chromosomal segments containing these markers provided a precise characterization of the amount of 7E chromosomal segments translocated. The association of these markers with BYDV resistance in various TLs suggested that the BYDVR locus is present in the long arm of chromosome 7E. This molecular characterization has formed a sound basis for further use of suitable translocation lines in targeted mapping of BYDV resistance and in developing elite wheat cultivars resistant to BYDV.

RECENT PUBLICATIONS

Dr. Joe Anderson has had two articles printed in AgriNews Publication, www.agrinews-pubs.com, May 25, 2007. Please check them out. The titles are "Virus now spreading across Indiana wheat fields" and "University lab requesting samples of infected wheat. Dr. Anderson submitted a picture of Resistant Wheat and Virus Susceptible Wheat. I'm glad he's doing the research. Look at the difference.

Barley Yellow Dwarf Virus Resistant Wheat

Barley Yellow Dwarf Virus Susceptible Wheat

Purdue University Small Grains program, and Ag Alumni Seeds and the USDA-ARS Small Grains Group described the various wheat and oat breeding and genetic research during the annual Wheat Field Day June 12 at the Purdue University Agronomy Center for Research and Education (ACRE). The producers and seedsmen were shown the progress that has occurred in developing elite wheat lines that contain higher levels of resistance to Fusarium head blight, septoria leaf and glume blotch, Hessian fly resistance and the new threats from stripe rust and stem rust. Many of these disease resistance traits have now been pyramided together to provide resistance to multiple pathogens. These lines are also high yielding wheat that produce flour with excellent baking characteristics. This field day also showed that barley yellow dwarf virus was extremely severe this year. Because of the mild fall weather in 2006, the winter wheat was heavily infected in the seedling stage with barley yellow dwarf virus. The only wheat lines that were able to combat this disease epidemic were those Purdue University-USDA lines that contain virus resistance genes. The difference between resistant lines and susceptible plants was very dramatic. The resistant plants were green and healthy looking, whereas those that were susceptible were yellow or brown and stunted. In addition to Indiana wheat producers, a group of wheat researchers from the University of Georgia were also at this wheat field day. This group which included faculty, postdoctoral fellows, graduate and undergraduate students, came to the field day as part of an NSF funded educational outreach to be able to see in action how a wheat genetics/breeding program functions to develop elite wheat varieties.

Refereed Journal Articles:

Anderson, JM, P Palukaitis, and M Zaitlin. 1992. A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants. Proc. Natl. Acad. Sci. (USA). 89:8759-8763.
Zaitlin, M, JM Anderson, KL Perry, L Zhang, and P Palukaitis. 1994. Specificity of replicase-mediated resistance to cucumber mosaic virus. Virology, 201:200- 205.
Nakata, PA., JM. Anderson, and TW. Okita. 1994. Structure and expression of the Potato ADP-glucose pyrophosphorylase small subunit. J. Biol. Chem. 269: 3 0798-30807.

Manuscripts Submitted:

Anderson, JM, DL Bucholtz, T Galli, and A Cook. 1997. Molecular and biochemical regulation of starch biosynthesis during barley seed development. (Plant Physiology).
Francki, MG, O Crasta, JM Anderson, H Sharma and HW Ohm. 1997. Structural organization of an alien Thinopyron intermedium group 7 chromosome in US soft red winter wheat (Triticum aestivum L.). (Genome)
Berzonski, WA, MG Francki, JM Anderson and HW Ohm. 1997. A cytological and molecular comparison of 1RS.1BL translocations in US soft red winter wheats (Triticum aestivum L.). (Euphytica)

Manuscripts In Preparation:

Anderson, JM, DL Bucholtz, A Greene, KL Perry, H Sharma, and H Ohm. 1997. Level and spectrum of resistance to barley yellow dwarf virus in a wheat line containing an alien wheatgrass (Thinopyron intermedium) group 7 chromosome. (For submission to Phytopathology)

ABSTRACTS

Anderson, JM, DL Bucholtz, T Galli, A Cook, and J Petik. 1995 Molecular and biochemical coordination of starch biosynthesis during barley seed development. Plant Physiol. 108S: 30.
Hodges, TK, R Aldemita, H Kononowicz-Hodges, B Macdonald, JM Anderson. 1996. Agrobacterium-mediated transformation of japonica and indica rice varieties. Workshop on In Vitro Manipulation of Wheat and Small Grains. June 22-27, World Congress on In Vitro Biology. San Francisco, CA.
Berzonski, WA, M Francki, JM Anderson and HW Ohm. 1996. A cytological and molecular comparison of soft red winter wheat varieties with 1RS.1BL. American Society of Agronomy Meeting, Nov. 3-8, Indianapolis, IN.
Crasta, O, M Francki, A Greene, DL Bucholtz, H Sharma, H Ohm and JM Anderson. 1996. Molecular Characterization of Thinopyrum chromatin in wheat: Toward targeting mapping of BYDV resistance. Second International Crop Science Congress. Nov. 17-23, New Delhi, India.
Anderson, J.M., O. Crasta, M. Francki, D. Bucholtz, H. Sharma and H.W. Ohm. Molecular and cytogenetic analysis of barley yellow dwarf virus resistant translocation lines containing Thinopyrum intermedium chromosomal segments. 1997. Plant Genome V. Jan. 12-17, San Diego, CA.