INDIANA
PURDUE UNIVERSITY
Departments of Agronomy, Entomology, and Botany and Plant Pathology, and the USDA-ARS, Purdue University, West Lafayette, IN 47907, USA.
J.M. Anderson, S.E. Cambron, C. Crane, S.B. Goodwin, S. Scofield, B. Schemerhorn, R.H. Shukle, and C.E. Williams (USDA-ARS); H.W. Ohm, M. Deb, L. Kong, and X. Shen (Department of Agronomy); G. Buechley, G. Shaner, and J.R. Xu (Department of Botany and Plant Pathology); and J. Stuart (Department of Entomology).
According to the USDA National Agricultural Statistics Service, Indiana farmers harvested 182,112 hectares (450,000 acres) of wheat in 2006, up 32% from 2005. Wheat yields in Indiana averaged 4,773 kg/ha (71 bu/a) in 2006, 1 bu less than the record high yield in 2005. Like most winters in Indiana since 1996, temperatures averaged above normal and winterkill due to low temperatures was limited. Similar to 2005, growing conditions for winter wheat in 2006 were excellent: ample soil moisture and cool temperatures continued to late June when much of the wheat crop was physiologically mature. Beginning in late June and through the harvest season to mid July, temperatures were elevated and soil moisture was limiting, providing excellent drying conditions during the harvest season, and resulting in high grain yields and high test weight. Acreage prospects for 2006-07: wet field conditions delayed harvest of corn and soybeans in September-October 2006, delaying wheat seeding, and likely reduced intended wheat area seeded.
Cool temperatures delayed the onset of symptoms of Fusarium head blight, although warm conditions beginning approximately two weeks after flowering in central to southern Indiana resulted in significant severity of the disease. Crop losses from other diseases, including powdery mildew, leaf rust, stem rust, Stagonospora glume blotch and Septoria leaf blotch were moderate to minor. Hessian fly was found in 2006 near Battleground, just north of Lafayette, IN. Evaluation with markers revealed that the population sample is similar to other populations collected in the upper Midwest and not similar to those from the Southeast.
INW0731, tested as P99608C1-1-3-4, was developed cooperatively by Purdue University and USDA-ARS and released in 2007. INW0731is a soft red winter wheat line and is the progeny of an F4 plant selection. The cultivar was performance tested at multiple locations in Indiana since 2004, in the 5-State regional nursery in 2005, the Uniform Eastern Winter Wheat nursery in 2006, and in the Preliminary Northern Uniform Winter Wheat Scab Nursery in 2005. INW0731has high yield potential, excellent soft wheat milling and baking quality, moderate resistance to Fusarium head blight (having resistance from Freedom and Fundulea 201R), moderate resistance to leaf rust, resistance/tolerance to yellow dwarf virus, powdery mildew, Stagonospora nodorum blotch, Septoria leaf blotch, soilborne mosaic virus, and wheat spindle streak mosaic virus, and is susceptible to Hessian fly, stripe rust and stem rust in Indiana. Adapted to southern Indiana and surrounding regions; INW0731 has survived winters very well in central and northern Indiana, but winters have been mild since 1996. The parentage of INW0731 is 'Sunset / Pioneer 2571 /3/ Clark // Roazon / Caldwell /4/ VPM / Moisson // Clark /3/ Clark*2 / Caldwell /9/ Caldwell*2 / S76 /8/ Beau*2 / Potomac // Auburn / Caldwell*2 /7/ Benhur / Arthur /6/ Laporte / Knox*2 /5/ Hart / Beau /4/Arthur /3/ Monon // Funo / Knox /10/ Freedom / Fundulea 201R'. After the last cross, plant selections were made in F2, F3, and F4, with the pedigree method of selection.
Characterization of plant processes manipulated by virulent Hessian fly (Christie Williams, Jill Nemacheck, Subhashree Subramanyam, Marcelo Giovanini, Kurt Saltzmann, and Stephen Baluch).
Oxidative burst. Resistant wheat was assayed for the rapid increase in the levels of active oxygen species, characteristic of an oxidative burst, as an early response to Hessian fly larvae. No correlation was found between Hessian fly larval feeding and accumulation of O2- or H2O2 in wheat tissues. Loss of resistance was not detected after treatments that inhibit NADPH oxidases and thus interrupt the signal pathway that generates an oxidative burst. In addition, Hessian fly larval feeding did not result in increased mRNA for genes that produce or scavenge active oxygen species. These data suggest that the wheat gene-for-gene recognition of Hessian fly larvae does not activate the oxidative burst component of resistance that is common to many gene-for-gene interactions between plants and other pathogens (Mol Plant Microbe Interact 19:1023-1033, 2006).
Putative feeding deterrent. Quantification of Hfr-3 mRNA, encoding a novel wheat germ agglutinin-like protein, in the incompatible interaction confirmed a rapid response up to 3,000-fold above the uninfested control. The abundance of mRNA was influenced by the number of larvae/plant, suggesting localized rather than systemic resistance, HFR-3 protein increased in parallel to the mRNA during incompatible interactions and was detected in both virulent and avirulent larvae, indicating ingestion. Antinutritional proteins, such as lectins, may be responsible for the apparent death by starvation of avirulent Hessian fly larvae during the initial few days of incompatible interactions with resistant wheat plants (Mol Plant Pathol 8:69-82, 2007).
Protein analysis. With collaborators, a standardized protein extraction protocol was developed that works for wheat as well as other monocot and dicot plants. This protocol significantly improves solubilization of total proteins. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer and subsequently solubilized with a modified O'Farrell lysis buffer. The separation of leaf total proteins by two-dimensional gel electrophoresis revealed improved solubilization and increased spot numbers, visualized with Coomassie brilliant blue staining (J Plant Biol 49:413-420, 2006).
Lab members. Subhashree Subramanyam is a Purdue University postdoctoral researcher. Kurt Saltzmann is a USDA-ARS postdoctoral researcher. Jill Nemacheck is a research technician.
Molecular interactions between the larval Hessian fly and wheat (Richard Shukle, Omprakash Mittapalli, Alisha Johnson, and Jacob Shreve).
Response of genes expressed in the larval Hessian fly during interactions with wheat. The focus of this work is to understand the molecular interactions that are induced or suppressed by Hessian fly larvae during their attack of wheat and that trigger host susceptibility or resistance. We have constructed a Hessian fly EST library and gene expression analyses have detected genes differentially expressed in larvae feeding on susceptible wheat compared to resistant wheat. Results have provided insight into the expression of genes involved in detoxification and antioxidant defense responses.
Comparative transcriptomics of larval salivary glands. Salivary gland EST libraries have been constructed for Hessian fly (USDA-ARS, Manhattan, KS), the Asian rice gall midge, and the orange wheat blossom midge (USDA-ARS, West Lafayette, IN). The discovery of transcripts in the larval salivary glands of all three of these gall midges that encode small secreted proteins supports the hypothesis that these secreted proteins are the elicitors that trigger host responses resulting in susceptibility or resistance. Comparative transcriptomics has identified novel genes encoding secreted proteins common to all three gall midges as well as novel genes encoding secreted proteins unique to each species. We speculate the genes in common encode proteins that have a common role in the parasitizing of host plants, while those unique to each species are involved in the adaptation of the gall midges to their respective host plant/tissue feeding site. Among the pools of the unique genes should reside genes for virulence.
RNAi knockdown to test the role of Hessian fly genes in parasitizing wheat. The role of Hessian fly genes identified through differential expression and/or comparative transcriptomics will be tested using small interfering RNAs (siRNAs) targeting specific transcripts. RNAi knockdown will initially be used to examine the role of three genes expressed in Hessian fly salivary gland that have been identified as encoding proteins in common with other gall midges and two genes novel to the Hessian fly.
Virus-induced gene silencing (VIGS). Using virus-induced gene silencing to identify genes required in disease resistance pathways of wheat (Amanda Brandt, Cahid Cakir, Megan Gillespie, and S. Scofield), we have developed a VIGS system, based on barley stripe mosaic virus, for the rapid analysis of gene function in hexaploid wheat. In VIGS, plants are infected with a virus that has been engineered to contain sequences from a plant gene of interest. The dsRNA produced as the virus replicates triggers the plant's sequence-specific RNA degradation mechanism, which targets all RNAs with homology to the viral genome for destruction. As the viral RNA contains transcribed plant sequence, any homologous host mRNAs also are targeted for destruction, resulting in silencing the expression of the plant gene of interest. This VIGS system has proven to be very effective in creating gene knockout phenotypes in hexaploid wheat and our lab is focusing on developing VIGS assays for the functional identification of genes required in the pathways providing resistance to leaf rust and Fusarium head blight.
Lab members. Amanda Brandt is a USDA-ARS research technician and Cahid Cakir is a USDA-ARS postdoctoral researcher.
Small Grain Cereal Virus detection (M. Deb and J.M. Anderson). In this study, a multiplex reverse transcription polymerase chain reaction (M-RT-PCR) method was developed for the simultaneous detection and discrimination of eight viruses including five strains of B/CYDVs, WSSMV, SBWMV, and WSMV. The protocol uses specific primers sets for each virus producing five B/CYDV distinct fragments for BYDV-PAV, BYDV-MAV, CYDV-RPV, and two unassigned Luteoviridae BYDV-SGV and -RMV, respectively. This system also produces WSSMV-, SBWMV-, and WSMV-specific amplicons, respectively. The eight amplicons produced in this one-tube PCR can be readily separated in a high resolution agarose gel. The amplification specificity of these primers was tested against a range of field samples from different parts of United States. This study has produced a rapid and specific wheat and small grain cereal virus diagnostic tool which will also be very effective in examining the epidemiology of these viral diseases.
Wheat–Thinopyrum mosaic chromosomes (K. Card, L. Ayala, N. Thompson, and J.M. Anderson). Previously, marker and virus inoculation analyses indicated that two lines that contained Thinopyrum-wheat translocations when crossed to Chinese Spring produced a large number of recombinants in which the translocation chromosomes consist of an array of wheat and Th. intermedium chromatin segments. From these recombinants a set of lines were identified that are resistant to B/CYDV and have interstitial Th. intermedium translocations. Current efforts are centered on characterizing the length of these translocations.
Identification of wheatgrass-specific molecular markers (E. Buescher and J.M. Anderson). Wheatgrass species such as Thinopyrum and Lophopyrum are important sources of useful traits particularly disease resistance to barley and cereal yellow dwarf viruses, leaf rust, and Fusarium head blight. Molecular markers derived from wheat typically do not identify wheatgrass-specific polymorphisms and consequently are used as negative (wheat DNA fragment missing) markers. In order to identify polymorphisms that are either codominant for wheatgrass and wheat or are dominant for wheatgrass, wheat oligonucleotide arrays were hybridized with RNA isolated from five wheat-wheatgrass substitution or addition lines each having a wheatgrass 7el1 or 7el2 chromosome and the reference wheat line Chinese Spring. These arrays were analyzed for SNPs and insertion/deletions using a robustified projection pursuit (RPP) algorithm (Cui et al. 2005). This analysis yielded approximately 44 putative 7E polymorphisms in addition to a large set of already known SNPs present in the wheat SNP database search (http://wheat.pw.usda.gov/GG2/blast.shtml). Current efforts are centered on validating the putative 7E polymorphisms through cloning and sequencing, mapping them to the 7E chromosomes for use as markers linked to wheatgrass-derived disease resistance traits.
Sequencing of Mycosphaerella graminicola (S.B. Goodwin). A project to sequence the genome of the Septoria tritici blotch pathogen, Mycosphaerella graminicola, was completed, and the 8.9x draft sequence (version 1) is available on the web pages of the Joint Genome Institute (http://genome.jgi-psf.org/Mycgr1/Mycgr1.home.html). A jamboree for manual annotation of the genome was convened in Walnut Creek, CA, 7-9 June, 2006, and included more than 20 participants. More than 1,200 genes have been annotated manually. The genome of approximately 40 Mb is being finished currently and contains 15 complete chromosome sequences from telomere to telomere and five more large scaffolds, four of which have a telomere at one end. Thus, the finished genome is expected to contain the 18 chromosomes predicted from the genetic linkage map. The few remaining gaps in the sequence correspond mostly to centromeres.
Research personnel. Don Huber, Department of Botany and Plant Pathology, retired in September 2006. Hari Sharma, Department of Agronomy, retired in December, 2006. Stephen Baluch is a Ph.D. student with co-advisors Christie Williams and Herb Ohm. Elizabeth Buescher is a Ph.D. student with co-advisors Joe Anderson and Herb Ohm. Megan Gillespie is a Ph.D. student with co-advisors Steve Scofield and Herb Ohm. Marcelo Giovanini with co-advisors Herb Ohm and Christie Williams, completed the Ph.D. degree and is in a postdoctoral position in his home country of Brazil. Julie Zwiesler-Vollick completed her postdoctoral position with Stephen Goodwin and accepted an Assistant Professor position at Lawrence Technical Institute in Detroit.