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, A. Johnson, J.A. Nemacheck, S. Scofield, B. Schemerhorn,
R.H. Shukle, and C.E. Williams (USDA-ARS); H.W. Ohm, M. Deb, L.
Kong, H.C. Sharma, and X. Shen (Department of Agronomy); G. Buechley,
D. Huber, 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 137,652 hectares (340,000 acres) of wheat in 2005, down from 440,000 acres in 2004 due to wet and rainy conditions during the wheat seeding season in the autuamn of 2004. Wheat yields in Indiana averaged 4,840 kg/ha (72 bu/acre) in 2005, a record, and up from 62 bu/acre in 2004. Like most winters in Indiana since 1996, temperatures averaged above normal and winterkill due to low temperatures was limited. Growing conditions for winter wheat in 2005 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 ideal drying conditions during the harvest season, and resulting in high grain yields and high test weight. Due to generally favorable wheat market prices and especially favorable seeding conditions during the autumn of 2005, area seeded to winter wheat for the 2006 harvest season was 460,000 acres, up 28 percent from area seeded in autumn 2004.
Cool temperatures limited the incidence and spread of Fusarium head blight but favored development of stripe rust, which was moderately severe in some fields in limited areas and for which some producers applied fungicides to limit crop damage. Crop losses from other diseases, including powdery mildew, leaf rust, stem rust, Stagonospora glume blotch, and Septoria leaf blotch were limited or negligible.
Using VIGS to identify genes required in disease resistance pathways of wheat (Amanda Brandt, Cahid Cakir, Lauren Grieg, and Steve Scofield). We have developed a virus-induced gene silencing 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 a range of wheat disease resistance pathways.
During the past year, we published our work using VIGS to demonstrate the requirement of the Lr21, RAR1, SGT1, and HSP90 genes for Lr21-mediated resistance to leaf rust (Scofield et al., 2005 Plant Physiol. 138: 2165-73). We have extended this analysis and are identifying other genes that are essential for Lr21-mediated resistance. Additionally, we have developed new VIGS assays that are being used to identify genes required for resistance to M. graminicola and F. graminearum.
Shortening the 7el2 segment that has FHB resistance (Xiaorong Shen, Hari Sharma, Lingrang Kong, and Herb Ohm). Robertsonian translocation T7DS·7el2L wheat line KS24-2 has FHB resistance gene(s) from Th. ponticum. The 7el2L likely has agronomic undesirable genes. KS24-2 was crossed to the ph1b mutant to facilitate homoeologous recombination. Among the ph1bph1b F2 plants, identified by marker WPG90, we selected translocation heterozygotes using dominant markers specific, respectively, for 7el2 and 7D. Plants in subsequent generations of self pollination were phenotyped for FHB resistance and genotyped with markers to identify recombinants with a shortened 7el2L segment. One F4 plant, 275-4, lost two marker loci, Xgwm333 and BE406148, but retained Xpsr129, BE445567 and Xcfa2240 on the distal part of the 7el2L segment. Thus, the amount of 7el2L chromatin was reduced while retaining the FHB resistance. This wheat line should be useful in breeding wheat for FHB resistance. The search for additional useful wheat lines with shortened 7el2L segment but that retain the FHB resistance is continuing.
Identifying virulence factors in Fusarium graminearum (Kyeyong Seong, Zhanming Hou, and Jin-Rong Xu). The REMI (Restriction-Enzyme Mediated Integration) approach was used to generate 11 pathogenicity mutants of F. graminearum, the causal agent of FHB (Seong et al. 2005b). Genetic analyses indicated that the defects in plant infection were tagged by the transforming vector in six of these mutants. In mutant M8, the transforming plasmid was integrated 110-bp upstream from the start codon of the cystathionine beta-lyase gene (CBL1). Genes disrupted by the transforming DNA in M68, M7, and M75 encoded a putative NADH: ubiquinone oxidoreductase, a beta-ZIP transcription factor, a transducin beta-subunit-like protein, respectively. In mutant 222, the transforming vector was inserted at amino acid 269 of the hydroxymethyl-glutaryl CoA reductase gene (HMR1) that encodes a key enzyme in sterol and isoprenoid biosynthesis. Further characterization revealed that the N-terminal portion of the HMR1 ORF has cryptic promoter activity (Seong et al. 2005a).
Host Resistance (Hathaithip Wiangjun and Joseph Anderson). The mechanism of intermediate wheatgrass (Th. intermedium)-derived CYDV resistance (Wiangjun and Anderson 2004, Phytopath 94:1102-1106) has been further elucidated. This resistance is clearly due to the reduced ability of the aphid to deposit virus into the phloem and a concomitant block of virus movement from the infection site when the virus is deposited into the vascular system. Further analyses have shown that callose deposition is not responsible for the block in virus movement within the sieve tubes. This resistance has two quite distinct components, suggesting that it will be a durable resistance.
Gene expression analysis (Mahua Deb, Bovaraghan Balaji, and J.M. Anderson). The expression patterns of 20 candidate defense-response genes in a susceptible and a resistant wheat line were examined at eight time points after infestation with nonviruliferous and viruliferous (BYDV-PAV/CYDV-RPV). These results indicate that some of these genes are both aphid and virus responsive. The susceptible line also shows a more pronounced gene-induction pattern than the resistant line for most genes although there are a few whose response is either repressed or enhanced in the viruliferous aphid-infested resistant line. This pathosytem, which has three components (aphid-virus-plant), clearly has a complicated defense-response pattern.
Wheat-Thinopyrum mosaic chromosomes (Katie Card, Ligia Ayala, Nicole Thompson, and J.M. Anderson). Previously, F2 progeny of two M4 lines crossed to Chinese Spring were examined with PCR markers for the presence of Th. intermedium segregating fragments. These data showed that a set of recombinants was identified that were a mosaic of wheat and Th. intermedium chromatin segments. New data have been obtained that now correlate this marker analysis with the presence or absence of the Bdv3 YDV resistance locus. These data have further identified several classes of recombinants in which the recombinant chromosome is primarily wheat yet the lines retain YDV resistance.
Chromosome segment 7E from Th. intermedium carrying yellow dwarf viruses resistance (H. Sharma, K. Card, J.M. Anderson, and H. Ohm). Research is in progress to shorten the 7E segment of wheat germ plasm line P961341 (Ohm et al. 2005) that has Bdv3 for resistance to yellow dwarf virus, via crossing P961341 to a ph1b mutant line and methods similar to those described above for 7el2L carrying resistance to Fusarium head blight.
Hessian fly/gall midge Transcriptomics (Richard H Shukle, Omprakash Mittapalli, and Alisha Johnson). We are revealing the catalog of mRNAs expressed in tissues of the larval Hessian fly during interactions with wheat, which will allow identification of genes or cellular pathways selectively turned on or off in response to extrinsic factors or intrinsic genetic programs. Additionally, we are cataloging mRNAs expressed in the midgut and salivary glands of the orange wheat blossom midge and the Asian rice gall midge. Identification of gene responses and a comparison of genes expressed between the Hessian fly, the wheat midge and the rice gall midge will provide for molecular dissection of the interactions with their respective host plants.
Assessing the phylogenetics of the Hessian fly using mitochondrial and nuclear markers (R. Shukle and A. Johnson). The phylogenetics and phylogeographic relationships of Hessian fly populations from Southwest Asia, the Mediterranean basin, and North America have been assessed using the mitochondrial 12S rRNA gene and a nuclear intron sequence from a Hessian fly white gene. Results have revealed that genotypes present in populations from Israel show the greatest genetic distance from the other populations, suggesting these genotypes are either ancestral or are an incipient species with Hessian fly. Phylogenetic analyses from the present study support the previously proposed hypothesis that Hessian fly dispersal in the Mediterranean basin proceeded from the Middle East/fertile crescent toward the western rim of the Mediterranean basin. Historical record and phylogenetic analyses suggest the genotypes in North America trace their ancestry to those present in the western rim of the Mediterranean basin. Analysis of molecular variance has revealed insight into variation within and between populations. These results are revealing new insight into the ancestry of Hessian fly in North America and into genetic variation in populations with respect to the appearance of virulent genotypes of the pest capable of surviving on formerly resistant wheat.
Characterization of plant processes manipulated by virulent Hessian fly (Christie Williams, Jill Nemacheck, Subhashree Subramanyam, Marcelo Giovanini, and Kurt Saltzmann). We demonstrated that increased protein abundance correlates with increased mRNA levels. Through cloning into expression vectors, protein purification and immuno-detection, we demonstrated that two wheat genes responsive to Hessian fly feeding were more abundant in incompatible interactions. These are the first proteins demonstrated to contribute to the Hessian fly-resistance response. A mechanism of resistance is suggested: because these are lectins, they probably function as feeding deterrents or to block absorption of nutrients in larval midgut. These antibodies will allow us to target cellular and organelle location in the plant and the larval midgut, and quantify expression on western blots. This work will determine whether components of resistance and susceptibility are cell-autonomous or systemic responses.
Up-regulation of both SAMDC and aminopropyl
transferase genes in susceptible plants implicates polyamine biosynthesis
as a pathway manipulated by the Hessian fly to benefit its development.
Up-regulation of a sorbitol transporter may supply a carbon source
while suppression of a lipid transporter gene may sequester resources
for use by developing larvae. These genes were also characterized
in wheat challenged with virus, chewing and sucking insects, and
abiotic stresses. This analysis distinguished genes specifically
involved in susceptibility to insects from general stress response
genes.
VIGS (virus induced gene silencing) verifies Hfr-2 gene
involvement in susceptibility. We demonstrated that the larvae
on silenced plants grew at a slower rate than larvae on control
plants, indicating that Hfr-2 gene activity is beneficial
to larval development. qRT-PCR confirmed that the gene was silenced.
This gene (up-regulated 800-fold in compatible interactions) encodes
a plant membrane pore-forming protein that probably allows delivery
of nutrients to developing larvae. Blocking this gene may provide
a novel form of resistance.
Developing Hessian fly larvae manipulate the amino acid content of wheat cells. Free amino acid analysis demonstrated increased plant production of five amino acids that cannot be synthesized by insects during compatible interactions. Most nonessential amino acids were not affected. This confirms our previous results showing that Round-up suppression of plant aromatic amino acids (essential to insect) resulted in larval death. This is the first evidence that Hf alters amino acid biosynthetic pathways of its host.
Development of microsatellite genetic markers in Hessian fly (Brandi Schemerhorn and Yan Ma Crane). Enriched microsatellite libraries were prepared from size-selected genomic DNA of Hessian fly. Approximately 81 % of the 52,224 recovered clones hybridized with microsatellite motif-specific probes. Of these, 8,256 clones were PCR screened, and 2,350 of them were successfully sequenced. Perfect microsatellites were contained in 55.8 % of the clones, and another 19.5 % contained at least one imperfect microsatellite. Polymorphism and reliability were tested in four Hessian fly biotypes, D, GP, O, and L, for 50 of the microsatellites in agarose gels. Twenty microsatellites were further tested with capillary electrophoresis. Of these, 17 behaved as a polymorphic single locus, two were invariant, and one represented a multiple locus. Preliminary results also indicate that there is no restriction in gene flow between the Hessian fly biotypes D, GP, O, and L.
Disease resistance (Jill Breeden, Ian Thompson, Emily Helliwell, and Stephen B. Goodwin). Real-time PCR was used to confirm differential expression of genes that probably are involved in the nonhost resistance response of barley to contact with the wheat pathogen M. graminicola. In previous experiments with the Affymetrix Barley1 genechip array, a large number of genes that were specifically up or down regulated in the non-host resistance response were identified. Real-time PCR confirmed the differential expression and also showed no change in control genes that did not change in the chip experiment. More than 30 of these genes had no useful annotations, yet appear to be involved in non-host resistance. Current work is involved with identifying putative functional domains within these genes.
In a collaborative project with Dr. Steve Scofield and his postdoc Cahid Cakir, we have been testing the effect of genes Sgt1, Rar1, and Hsp90 on the expression of resistance genes Stb2 and Stb4 against M. graminicola. The VIGS technique that was developed in the Scofield lab was used to silence expression of the three genes listed above to test for a change in phenotype from resistant to susceptible. The initial experiments looked promising and a change in phenotype was seen for resistance gene Stb4. Those experiments are being repeated with increased replications to confirm whether the observed change is real.
Fungal genetics (Jessica Cavaletto and S.B. Goodwin). A project to sequence the genome of the septoria tritici blotch pathogen, Mycosphaerella graminicola, was begun in collaboration with Dr. Gert Kema and other scientists at Plant Research International in Wageningen, the Netherlands, through the Community Sequencing Program at the D.O.E.-Joint Genome Institute. The genome was sequenced to almost 9x coverage and the initial assembly was completed during November of 2005. The genome size is estimated to be 41.8 Mb, slightly larger than thought previously. In addition, a draft mitochondrial assembly of 43,962 bases is available. Over 500,000 sequence reads from the project have been deposited and are available through GenBank. Machine annotation of the genomic sequence is now in progress and plans for a community-wide annotation effort culminating in an annotation jamboree that will be open to all interested participants is anticipated for 2006.
Final work on the genetics of microsatellites
in M. graminicola was completed and 23 loci were
added to the existing genetic linkage map. These markers are highly
polymorphic and are available for analyses of fungal genetic and
population biology.
Katie Card and Danielle Posch have joined the Anderson lab as Biological Science Research Technicians. Katie is in charge of developing and utilizing DNA markers linked to the yellow dwarf virus resistance locus, Bdv3 and additional YDV resistance loci derived from Thinopyrum. Danielle is primarily responsible for developing oat DNA markers and will be constructing SSR enriched libraries for identifying polymorphic SSRs for mapping traits in oats. Emily Helliwell joined the Goodwin lab as a Masters student during the autumn of 2005. Emily is interested in marker-assisted selection and will be working on developing new markers that are linked to the Stb2 gene for resistance to M. graminicola that is located on chromosome 3BS. Dr. Ian Thompson joined the Goodwin lab as a Biological Science Research Technician during the autumn of 2006. Ian has a Ph.D. in Plant Pathology from Purdue University and is in charge of greenhouse testing and marker analysis. He has several ongoing projects, including backcrossing various STB resistance genes into common susceptible genetic backgrounds, introgressing a gene for resistance to STB from Thinopyrum into wheat, and developing improved methods for identifying and analyzing quantitative resistance to STB in wheat. Dr. Julie Zwiesler-Vollick joined the Goodwin lab as a postdoctoral associate during July of 2005. Julie received a Ph.D. in genetics from the DOE-Plant Research Laboratory at Michigan State University under the direction of Dr. Sheng-Yang He, and had two years of postdoctoral experience with Dr. Anne Osbourn at the Sainsbury Laboratory in Norwich, England, before coming to West Lafayette. She has funding from the National Science Foundation to work on non-model systems, and is analyzing secreted proteins from wheat and Mycosphaerella graminicola within apoplastic fluids to understand the molecular basis for host-pathogen interactions. She also has a side project to clone and analyze fungal mating-type sequences. Jill Breeden left the Goodwin lab and moved on to a position with the U.S. Forest Service. Brett Ochs joined the Ohm lab studying for the Ph.D. degree; his thesis research is focused on genetics and mapping of resistance to Stagonospora glume blotch resistance. Kristen Rinehart joined the Ohm lab studying for the MS degree; her thesis research is focused on genetics and mapping of a new source of resistance to Hessian fly. Paul Werner joined the Ohm lab studying for the Ph.D. degree; his thesis research is focused on genetics and mapping of resistance to yellow dwarf virus and slow crown rusting resistance in oat. Dr. Kurt Saltzmann joined the Williams lab early in 2006, after graduating from the Purdue department of Entomology. He is identifying and characterizing the expression of wheat genes that respond to avirulent Hessian fly larvae, and has identified wheat amino acids that become more abundant during compatible interactions. Dr. Nagesh Sardesai has moved across the street to the lab of Dr. Stan Gelvin where he is now working on Arabidopsis.