Publications by authors named "Scot H Hulbert"

31 Publications

Image-Based Phenotyping of Flowering Intensity in Cool-Season Crops.

Sensors (Basel) 2020 Mar 6;20(5). Epub 2020 Mar 6.

Department of Biological Systems Engineering, Washington State University, Pullman, WA 99164, USA.

The timing and duration of flowering are key agronomic traits that are often associated with the ability of a variety to escape abiotic stress such as heat and drought. Flowering information is valuable in both plant breeding and agricultural production management. Visual assessment, the standard protocol used for phenotyping flowering, is a low-throughput and subjective method. In this study, we evaluated multiple imaging sensors (RGB and multiple multispectral cameras), image resolution (proximal/remote sensing at 1.6 to 30 m above ground level/AGL), and image processing (standard and unsupervised learning) techniques in monitoring flowering intensity of four cool-season crops (canola, camelina, chickpea, and pea) to enhance the accuracy and efficiency in quantifying flowering traits. The features (flower area, percentage of flower area with respect to canopy area) extracted from proximal (1.6-2.2 m AGL) RGB and multispectral (with near infrared, green and blue band) image data were strongly correlated ( up to 0.89) with visual rating scores, especially in pea and canola. The features extracted from unmanned aerial vehicle integrated RGB image data (15-30 m AGL) could also accurately detect and quantify large flowers of winter canola ( up to 0.84), spring canola ( up to 0.72), and pea ( up to 0.72), but not camelina or chickpea flowers. When standard image processing using thresholds and unsupervised machine learning such as k-means clustering were utilized for flower detection and feature extraction, the results were comparable. In general, for applicability of imaging for flower detection, it is recommended that the image data resolution (i.e., ground sampling distance) is at least 2-3 times smaller than that of the flower size. Overall, this study demonstrates the feasibility of utilizing imaging for monitoring flowering intensity in multiple varieties of evaluated crops.
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http://dx.doi.org/10.3390/s20051450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7085647PMC
March 2020

Analysis of miRNAs in Two Wheat Cultivars Infected With f. sp. .

Front Plant Sci 2019 10;10:1574. Epub 2020 Jan 10.

Department of Plant Pathology, Washington State University, Pullman, WA, United States.

MicroRNAs are small RNAs that regulate gene expression in eukaryotes. In this study, we analyzed the small RNA profiles of two cultivars that exhibit different reactions to stripe rust infection: one susceptible, the other partially resistant. Using small RNA libraries prepared from the two wheat cultivars infected with stripe rust fungus ( f. sp. ), we identified 182 previously known miRNAs, 91 variants of known miRNAs, and 163 candidate novel wheat miRNAs. Known miRNA loci were usually copied in all three wheat sub-genomes, whereas novel miRNA loci were often specific to a single sub-genome. DESeq2 analysis of differentially expressed microRNAs revealed 23 miRNAs that exhibit cultivar-specific differences. TA078/miR399b showed cultivar-specific differential regulation in response to infection. Using different target prediction algorithms, 145 miRNAs were predicted to target wheat genes, while 69 miRNAs were predicted to target fungal genes. We also confirmed reciprocal expression of TA078/miR399b and tae-miR9664 and their target genes in different treatments, providing evidence for miRNA-mediated regulation during infection. Both known and novel miRNAs were predicted to target fungal genes, suggesting trans-kingdom regulation of gene expression. Overall, this study contributes to the current repository of wheat miRNAs and provides novel information on the yet-uncharacterized roles for miRNAs in the wheat-stripe rust pathosystem.
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http://dx.doi.org/10.3389/fpls.2019.01574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965360PMC
January 2020

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils.

J Vis Exp 2019 02 9(144). Epub 2019 Feb 9.

Department of Crop and Soil Sciences, Washington State University;

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.
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http://dx.doi.org/10.3791/59137DOI Listing
February 2019

A novel fungal effector from Puccinia graminis suppressing RNA silencing and plant defense responses.

New Phytol 2019 05 13;222(3):1561-1572. Epub 2019 Feb 13.

Department of Plant Pathology, Washington State University, Pullman, WA, 99164-6430, USA.

Fungal plant pathogens, like rust-causing biotrophic fungi, secrete hundreds of effectors into plant cells to subvert host immunity and promote pathogenicity on their host plants by manipulating specific physiological processes or signal pathways, but the actual function has been demonstrated for very few of these proteins. Here, we show that the PgtSR1 effector proteins, encoded by two allelic genes (PgtSR1-a and PgtSR1-b), from the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt), suppress RNA silencing in plants and impede plant defenses by altering the abundance of small RNAs that serve as defense regulators. Expression of the PgtSR1s in plants revealed that the PgtSR1s promote susceptibility to multiple pathogens and partially suppress cell death triggered by multiple R proteins. Overall, our study provides the first evidence that the filamentous fungus P. graminis has evolved to produce fungal suppressors of RNA silencing and indicates that PgtSR1s suppress both basal defenses and effector triggered immunity.
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http://dx.doi.org/10.1111/nph.15676DOI Listing
May 2019

Genomic insights into host adaptation between the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) and the barley stripe rust pathogen (Puccinia striiformis f. sp. hordei).

BMC Genomics 2018 Sep 12;19(1):664. Epub 2018 Sep 12.

Department of Plant Pathology, Washington State University, Pullman, WA, 99164-6430, USA.

Background: Plant fungal pathogens can rapidly evolve and adapt to new environmental conditions in response to sudden changes of host populations in agro-ecosystems. However, the genomic basis of their host adaptation, especially at the forma specialis level, remains unclear.

Results: We sequenced two isolates each representing Puccinia striiformis f. sp. tritici (Pst) and P. striiformis f. sp. hordei (Psh), different formae speciales of the stripe rust fungus P. striiformis highly adapted to wheat and barley, respectively. The divergence of Pst and Psh, estimated to start 8.12 million years ago, has been driven by high nucleotide mutation rates. The high genomic variation within dikaryotic urediniospores of P. striiformis has provided raw genetic materials for genome evolution. No specific gene families have enriched in either isolate, but extensive gene loss events have occurred in both Pst and Psh after the divergence from their most recent common ancestor. A large number of isolate-specific genes were identified, with unique genomic features compared to the conserved genes, including 1) significantly shorter in length; 2) significantly less expressed; 3) significantly closer to transposable elements; and 4) redundant in pathways. The presence of specific genes in one isolate (or forma specialis) was resulted from the loss of the homologues in the other isolate (or forma specialis) by the replacements of transposable elements or losses of genomic fragments. In addition, different patterns and numbers of telomeric repeats were observed between the isolates.

Conclusions: Host adaptation of P. striiformis at the forma specialis level is a complex pathogenic trait, involving not only virulence-related genes but also other genes. Gene loss, which might be adaptive and driven by transposable element activities, provides genomic basis for host adaptation of different formae speciales of P. striiformis.
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http://dx.doi.org/10.1186/s12864-018-5041-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134786PMC
September 2018

Host-Induced Gene Silencing (HIGS) for Elucidating Puccinia Gene Function in Wheat.

Methods Mol Biol 2018 ;1848:139-150

Department of Plant Pathology, Washington State University, Pullman, WA, USA.

Biotrophic fungi (Puccinia spp.) cause devastating diseases of wheat and other cereal species globally. The function of large repertories of genes from Puccinia spp. still needs to be discovered to understand the infection process of these obligate parasites, eventually to protect plants from rust diseases. Functional analysis of targeted genes is challenging due to the inherent difficulties with culturing the fungus and transforming the host. RNA interference (RNAi) is a conserved gene regulation process in eukaryotes and known to be a powerful genetic tool in plant biotechnology. More recently, host-induced gene silencing (HIGS) has been developed to assess pathogen gene function in plants. HIGS is an RNAi-based process where double stranded RNA (dsRNA) homologous to a pathogen gene can be expressed in a plant to induce targeted silencing of the pathogen gene. Here we described a detailed HIGS protocol for functional analysis of rust genes from Puccinia species in cereals. As an example we describe an experiment silencing the tryptophan 2-monooxygenase gene (Pgt-IaaM) from Puccinia graminis f. sp. tritici (Pgt) that is involved in virulence to wheat.
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http://dx.doi.org/10.1007/978-1-4939-8724-5_12DOI Listing
May 2019

Genome Sequence Resources for the Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) and the Barley Stripe Rust Pathogen (Puccinia striiformis f. sp. hordei).

Mol Plant Microbe Interact 2018 11 7;31(11):1117-1120. Epub 2018 Sep 7.

1 Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, U.S.A.

Puccinia striiformis f. sp. tritici causes devastating stripe (yellow) rust on wheat and P. striiformis f. sp. hordei causes stripe rust on barley. Several P. striiformis f. sp. tritici genomes are available, but no P. striiformis f. sp. hordei genome is available. More genomes of P. striiformis f. sp. tritici and P. striiformis f. sp. hordei are needed to understand the genome evolution and molecular mechanisms of their pathogenicity. We sequenced P. striiformis f. sp. tritici isolate 93-210 and P. striiformis f. sp. hordei isolate 93TX-2, using PacBio and Illumina technologies and RNA sequencing. Their genomic sequences were assembled to contigs with high continuity and showed significant structural differences. The circular mitochondria genomes of both were complete. These genomes provide high-quality resources for deciphering the genomic basis of rapid evolution and host adaptation, identifying genes for avirulence and other important traits, and studying host-pathogen interactions.
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http://dx.doi.org/10.1094/MPMI-04-18-0107-ADOI Listing
November 2018

Analysis of Extreme Phenotype Bulk Copy Number Variation (XP-CNV) Identified the Association of with Resistance to Goss's Wilt of Maize.

Front Plant Sci 2018 9;9:110. Epub 2018 Feb 9.

Department of Plant Pathology, Kansas State University, Manhattan, KS, United States.

Goss's wilt (GW) of maize is caused by the Gram-positive bacterium subsp. (Cmn) and has spread in recent years throughout the Great Plains, posing a threat to production. The genetic basis of plant resistance is unknown. Here, a simple method for quantifying disease symptoms was developed and used to select cohorts of highly resistant and highly susceptible lines known as extreme phenotypes (XP). Copy number variation (CNV) analyses using whole genome sequences of bulked XP revealed 141 genes containing CNV between the two XP groups. The CNV genes include the previously identified common rust resistant locus . Multiple accessions with distinct haplotypes in an otherwise susceptible accession exhibited hypersensitive responses upon inoculation. GW provides an excellent system for the genetic dissection of diseases caused by closely related subspecies of . Further work will facilitate breeding strategies to control GW and provide needed insight into the resistance mechanism of important related diseases such as bacterial canker of tomato and bacterial ring rot of potato.
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http://dx.doi.org/10.3389/fpls.2018.00110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812337PMC
February 2018

Community Structure, Species Variation, and Potential Functions of Rhizosphere-Associated Bacteria of Different Winter Wheat () Cultivars.

Front Plant Sci 2017 13;8:132. Epub 2017 Feb 13.

Department of Plant Pathology, Washington State University, Pullman WA, USA.

Minimal tillage management of extensive crops like wheat can provide significant environmental services but can also lead to adverse interactions between soil borne microbes and the host. Little is known about the ability of the wheat cultivar to alter the microbial community from a long-term recruitment standpoint, and whether this recruitment is consistent across field sites. To address this, nine winter wheat cultivars were grown for two consecutive seasons on the same plots on two different farm sites and assessed for their ability to alter the rhizosphere bacterial communities in a minimal tillage system. Using deep amplicon sequencing of the V1-V3 region of the 16S rDNA, a total of 26,604 operational taxonomic units (OTUs) were found across these two sites. A core bacteriome consisting of 962 OTUs were found to exist in 95% of the wheat rhizosphere samples. Differences in the relative abundances for these wheat cultivars were observed. Of these differences, 24 of the OTUs were found to be significantly different by wheat cultivar and these differences occurred at both locations. Several of the cultivar-associated OTUs were found to correspond with strains that may provide beneficial services to the host plant. Network correlations demonstrated significant co-occurrences for different taxa and their respective OTUs, and in some cases, these interactions were determined by the wheat cultivar. Microbial abundances did not play a role in the number of correlations, and the majority of the co-occurrences were shown to be positively associated. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States was used to determine potential functions associated with OTUs by association with rhizosphere members which have sequenced metagenomics data. Potentially beneficial pathways for nitrogen, sulfur, phosphorus, and malate metabolism, as well as antimicrobial compounds, were inferred from this analysis. Differences in these pathways and their associated functions were found to differ by wheat cultivar. In conclusion, our study suggests wheat cultivars are involved in shaping the rhizosphere by differentially altering the bacterial OTUs consistently across different sites, and these altered bacterial communities may provide beneficial services to the host.
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http://dx.doi.org/10.3389/fpls.2017.00132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303725PMC
February 2017

Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses.

Phytopathology 2017 Jan 18;107(1):75-83. Epub 2016 Oct 18.

First, second, fourth, fifth, and seventh authors: Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430; and third and sixth authors: Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339.

Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector-R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS-2, and GPA/RBP-1) and one mutated R gene-Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.
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http://dx.doi.org/10.1094/PHYTO-02-16-0083-RDOI Listing
January 2017

Small RNAs from the wheat stripe rust fungus (Puccinia striiformis f.sp. tritici).

BMC Genomics 2015 Sep 21;16:718. Epub 2015 Sep 21.

Molecular Plant Sciences, Washington State University, Pullman, WA, USA.

Background: Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is a costly global disease that burdens farmers with yield loss and high fungicide expenses. This sophisticated biotrophic parasite infiltrates wheat leaves and develops infection structures inside host cells, appropriating nutrients while suppressing the plant defense response. Development in most eukaryotes is regulated by small RNA molecules, and the success of host-induced gene silencing technology in Puccinia spp. implies the existence of a functional RNAi system. However, some fungi lack this capability, and small RNAs have not yet been reported in rust fungi. The objective of this study was to determine whether P. striiformis carries an endogenous small RNA repertoire.

Results: We extracted small RNA from rust-infected wheat flag leaves and performed high-throughput sequencing. Two wheat cultivars were analyzed: one is susceptible; the other displays partial high-temperature adult plant resistance. Fungal-specific reads were identified by mapping to the P. striiformis draft genome and removing reads present in uninfected control libraries. Sequencing and bioinformatics results were verified by RT-PCR. Like other RNAi-equipped fungi, P. striiformis produces large numbers of 20-22 nt sequences with a preference for uracil at the 5' position. Precise post-transcriptional processing and high accumulation of specific sRNA sequences were observed. Some predicted sRNA precursors possess a microRNA-like stem-loop secondary structure; others originate from much longer inverted repeats containing gene sequences. Finally, sRNA-target prediction algorithms were used to obtain a list of putative gene targets in both organisms. Predicted fungal target genes were enriched for kinases and small secreted proteins, while the list of wheat targets included homologs of known plant resistance genes.

Conclusions: This work provides an inventory of small RNAs endogenous to an important plant pathogen, enabling further exploration of gene regulation on both sides of the host/parasite interaction. We conclude that small RNAs are likely to play a role in regulating the complex developmental processes involved in stripe rust pathogenicity.
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http://dx.doi.org/10.1186/s12864-015-1895-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4578785PMC
September 2015

Pushing the boundaries of resistance: insights from Brachypodium-rust interactions.

Front Plant Sci 2015 30;6:558. Epub 2015 Jul 30.

Department of Plant Pathology, University of Minnesota , St. Paul, MN, USA ; Stakman-Borlaug Center for Sustainable Plant Health, University of Minnesota , St. Paul, MN, USA.

The implications of global population growth urge transformation of current food and bioenergy production systems to sustainability. Members of the family Poaceae are of particular importance both in food security and for their applications as biofuel substrates. For centuries, rust fungi have threatened the production of valuable crops such as wheat, barley, oat, and other small grains; similarly, biofuel crops can also be susceptible to these pathogens. Emerging rust pathogenic races with increased virulence and recurrent rust epidemics around the world point out the vulnerability of monocultures. Basic research in plant immunity, especially in model plants, can make contributions to understanding plant resistance mechanisms and improve disease management strategies. The development of the grass Brachypodium distachyon as a genetically tractable model for monocots, especially temperate cereals and grasses, offers the possibility to overcome the experimental challenges presented by the genetic and genomic complexities of economically valuable crop plants. The numerous resources and tools available in Brachypodium have opened new doors to investigate the underlying molecular and genetic bases of plant-microbe interactions in grasses and evidence demonstrating the applicability and advantages of working with B. distachyon is increasing. Importantly, several interactions between B. distachyon and devastating plant pathogens, such rust fungi, have been examined in the context of non-host resistance. Here, we discuss the use of B. distachyon in these various pathosystems. Exploiting B. distachyon to understand the mechanisms underpinning disease resistance to non-adapted rust fungi may provide effective and durable approaches to fend off these pathogens. The close phylogenetic relationship among Brachypodium spp. and grasses with industrial and agronomic value support harnessing this model plant to improve cropping systems and encourage its use in translational research.
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http://dx.doi.org/10.3389/fpls.2015.00558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519692PMC
August 2015

Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia.

BMC Genomics 2015 Aug 5;16:579. Epub 2015 Aug 5.

Department of Plant Pathology, Washington State University, Pullman, WA, 99164-6430, USA.

Background: The cereal rust fungi are destructive pathogens that affect grain production worldwide. Although the genomic and transcript sequences for three Puccinia species that attack wheat have been released, the functions of large repertories of genes from Puccinia still need to be addressed to understand the infection process of these obligate parasites. Host-induced gene silencing (HIGS) has emerged a useful tool to examine the importance of rust fungus genes while growing within host plants. In this study, HIGS was used to test genes from Puccinia with transcripts enriched in haustoria for their ability to interfere with full development of the rust fungi.

Results: Approximately 1200 haustoria enriched genes from Puccinia graminis f. sp. tritici (Pgt) were identified by comparative RNA sequencing. Virus-induced gene silencing (VIGS) constructs with fragments of 86 Puccinia genes, were tested for their ability to interfere with full development of these rust fungi. Most of the genes tested had no noticeable effects, but 10 reduced Pgt development after co-inoculation with the gene VIGS constructs and Pgt. These included a predicted glycolytic enzyme, two other proteins that are probably secreted and involved in carbohydrate or sugar metabolism, a protein involved in thiazol biosynthesis, a protein involved in auxin biosynthesis, an amino acid permease, two hypothetical proteins with no conserved domains, a predicted small secreted protein and another protein predicted to be secreted with similarity to bacterial proteins involved in membrane transport. Transient silencing of four of these genes reduced development of P. striiformis (Pst), and three of also caused reduction of P. triticina (Pt) development.

Conclusions: Partial suppression of transcripts involved in a large variety of biological processes in haustoria cells of Puccinia rusts can disrupt their development. Silencing of three genes resulted in suppression of all three rust diseases indicating that it may be possible to engineer durable resistance to multiple rust pathogens with a single gene in transgenic wheat plants for sustainable control of cereal rusts.
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http://dx.doi.org/10.1186/s12864-015-1791-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524123PMC
August 2015

Pyrosequencing reveals the predominance of pseudomonadaceae in gut microbiome of a gall midge.

Pathogens 2014 Jun 11;3(2):459-72. Epub 2014 Jun 11.

Department of Entomology, Kansas State University, Manhattan, KS 66506, USA.

Gut microbes are known to play various roles in insects such as digestion of inaccessible nutrients, synthesis of deficient amino acids, and interaction with ecological environments, including host plants. Here, we analyzed the gut microbiome in Hessian fly, a serious pest of wheat. A total of 3,654 high quality sequences of the V3 hypervariable region of the 16S rRNA gene were obtained through 454-pyrosequencing. From these sequences, 311 operational taxonomic units (OTUs) were obtained at the >97% similarity cutoff. In the gut of 1st instar, otu01, a member of Pseudomonas, was predominant, representing 90.2% of total sequences. otu13, an unidentified genus in the Pseudomonadaceae family, represented 1.9% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 2nd instar, otu01 and otu13 decreased to 85.5% and 1.5%, respectively. otu04, a member of Buttiauxella, represented 9.7% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 3rd instar, otu01 and otu13 further decreased to 29.0% and 0%, respectively. otu06, otu08, and otu16, also three members of the Pseudomonadaceae family were 13.2%, 8.6%, and 2.3%, respectively. In addition, otu04 and otu14, two members of the Enterobacteriaceae family, were 4.7% and 2.5%; otu18 and otu20, two members of the Xanthomonadaceae family, were 1.3% and 1.2%, respectively; otu12, a member of Achromobacter, was 4.2%; otu19, a member of Undibacterium, was 1.4%; and otu9, otu10, and otu15, members of various families, were 6.1%, 6.3%, and 1.9%, respectively. The investigation into dynamics of Pseudomonas, the most abundant genera, revealed that its population level was at peak in freshly hatched or 1 day larvae as well as in later developmental stages, thus suggesting a prominent role for this bacterium in Hessian fly development and in its interaction with host plants. This study is the first comprehensive survey on bacteria associated with the gut of a gall midge, and provides a foundation for future studies to elucidate the roles of gut microbes in Hessian fly virulence and biology.
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http://dx.doi.org/10.3390/pathogens3020459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243456PMC
June 2014

Characterization of a tryptophan 2-monooxygenase gene from Puccinia graminis f. sp. tritici involved in auxin biosynthesis and rust pathogenicity.

Mol Plant Microbe Interact 2014 Mar;27(3):227-35

The plant hormone indole-3-acetic acid (IAA) is best known as a regulator of plant growth and development but its production can also affect plant-microbe interactions. Microorganisms, including numerous plant-associated bacteria and several fungi, are also capable of producing IAA. The stem rust fungus Puccinia graminis f. sp. tritici induced wheat plants to accumulate auxin in infected leaf tissue. A gene (Pgt-IaaM) encoding a putative tryptophan 2-monooxygenase, which makes the auxin precursor indole-3-acetamide (IAM), was identified in the P. graminis f. sp. tritici genome and found to be expressed in haustoria cells in infected plant tissue. Transient silencing of the gene in infected wheat plants indicated that it was required for full pathogenicity. Expression of Pgt-IaaM in Arabidopsis caused a typical auxin expression phenotype and promoted susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000.
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http://dx.doi.org/10.1094/MPMI-09-13-0289-FIDOI Listing
March 2014

Role of bacterial communities in the natural suppression of Rhizoctonia solani bare patch disease of wheat (Triticum aestivum L.).

Appl Environ Microbiol 2013 Dec 20;79(23):7428-38. Epub 2013 Sep 20.

Department of Plant Pathology, Washington State University, Pullman, Washington, USA.

Rhizoctonia bare patch and root rot disease of wheat, caused by Rhizoctonia solani AG-8, develops as distinct patches of stunted plants and limits the yield of direct-seeded (no-till) wheat in the Pacific Northwest of the United States. At the site of a long-term cropping systems study near Ritzville, WA, a decline in Rhizoctonia patch disease was observed over an 11-year period. Bacterial communities from bulk and rhizosphere soil of plants from inside the patches, outside the patches, and recovered patches were analyzed by using pyrosequencing with primers designed for 16S rRNA. Taxa in the class Acidobacteria and the genus Gemmatimonas were found at higher frequencies in the rhizosphere of healthy plants outside the patches than in that of diseased plants from inside the patches. Dyella and Acidobacteria subgroup Gp7 were found at higher frequencies in recovered patches. Chitinophaga, Pedobacter, Oxalobacteriaceae (Duganella and Massilia), and Chyseobacterium were found at higher frequencies in the rhizosphere of diseased plants from inside the patches. For selected taxa, trends were validated by quantitative PCR (qPCR), and observed shifts of frequencies in the rhizosphere over time were duplicated in cycling experiments in the greenhouse that involved successive plantings of wheat in Rhizoctonia-inoculated soil. Chryseobacterium soldanellicola was isolated from the rhizosphere inside the patches and exhibited significant antagonism against R. solani AG-8 in vitro and in greenhouse tests. In conclusion, we identified novel bacterial taxa that respond to conditions affecting bare patch disease symptoms and that may be involved in suppression of Rhizoctonia root rot and bare batch disease.
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http://dx.doi.org/10.1128/AEM.01610-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837727PMC
December 2013

Genomic and resistance gene homolog diversity of the dominant tallgrass prairie species across the U.S. Great Plains precipitation gradient.

PLoS One 2011 Apr 12;6(4):e17641. Epub 2011 Apr 12.

Department of Plant Pathology, Kansas State University, Manhattan, Kansas, United States of America.

Background: Environmental variables such as moisture availability are often important in determining species prevalence and intraspecific diversity. The population genetic structure of dominant plant species in response to a cline of these variables has rarely been addressed. We evaluated the spatial genetic structure and diversity of Andropogon gerardii populations across the U.S. Great Plains precipitation gradient, ranging from approximately 48 cm/year to 105 cm/year.

Methodology/principal Findings: Genomic diversity was evaluated with AFLP markers and diversity of a disease resistance gene homolog was evaluated by PCR-amplification and digestion with restriction enzymes. We determined the degree of spatial genetic structure using Mantel tests. Genomic and resistance gene homolog diversity were evaluated across prairies using Shannon's index and by averaging haplotype dissimilarity. Trends in diversity across prairies were determined using linear regression of diversity on average precipitation for each prairie. We identified significant spatial genetic structure, with genomic similarity decreasing as a function of distance between samples. However, our data indicated that genome-wide diversity did not vary consistently across the precipitation gradient. In contrast, we found that disease resistance gene homolog diversity was positively correlated with precipitation.

Significance: Prairie remnants differ in the genetic resources they maintain. Selection and evolution in this disease resistance homolog is environmentally dependent. Overall, we found that, though this environmental gradient may not predict genomic diversity, individual traits such as disease resistance genes may vary significantly.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017641PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075248PMC
April 2011

Development of a host-induced RNAi system in the wheat stripe rust fungus Puccinia striiformis f. sp. tritici.

Mol Plant Microbe Interact 2011 May;24(5):554-61

Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA.

Rust fungi cause devastating diseases of wheat and other cereal species globally. Genetic resistance is the preferred method to control rusts but the effectiveness of race-specific resistance is typically transient due to the genetic plasticity of rust populations. The advent of RNA interference (RNAi) technology has shown promise for the engineering of resistance to some biotrophic pathogens in plants by altering the expression of essential pathogens' genes. Gene fragments from the rust fungi Puccinia striiformis f. sp. tritici or P. graminis f. sp. tritici were delivered to plant cells through the Barley stripe mosaic virus system, and some reduced the expression of the corresponding genes in the rust fungus. The ability to detect suppression was associated with the expression patterns of the fungal genes because reduction was only detected in transcripts with relatively high levels of expression in fungal haustoria. The results indicate that an in planta RNAi approach can be used in functional genomics research for rust fungi and that it could potentially be used to engineer durable resistance.
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http://dx.doi.org/10.1094/MPMI-10-10-0229DOI Listing
May 2011

Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes.

Mol Genet Genomics 2010 Jun 5;283(6):591-602. Epub 2010 May 5.

Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274, USA.

Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant.
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http://dx.doi.org/10.1007/s00438-010-0536-5DOI Listing
June 2010

Identification of a maize locus that modulates the hypersensitive defense response, using mutant-assisted gene identification and characterization.

Genetics 2010 Mar 22;184(3):813-25. Epub 2010 Feb 22.

Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907-2054, USA.

Potentially useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. In this study, a novel genetic technique called Mutant-Assisted Gene Identification and Characterization is used to identify naturally occurring loci modulating the hypersensitive defense response (HR) in maize. Mutant-Assisted Gene Identification and Characterization facilitates the identification of naturally occurring alleles underlying phenotypic variation from diverse germplasm, using a mutant phenotype as a "reporter." In this study the reporter phenotype was caused by a partially dominant autoactive disease resistance gene, Rp1-D21, which caused HR lesions to form spontaneously all over the plant. Here it is demonstrated that the Rp1-D21 phenotype is profoundly affected by genetic background. By crossing the Rp1-D21 gene into the IBM mapping population, it was possible to map and identify Hrml1 on chromosome 10, a locus responsible for modulating the HR phenotype conferred by Rp1-D21. Other loci with smaller effects were identified on chromosomes 1 and 9. These results demonstrate that Mutant-Assisted Gene Identification and Characterization is a viable approach for identifying naturally occurring useful genetic variation.
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http://dx.doi.org/10.1534/genetics.109.111880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845348PMC
March 2010

Generation and analysis of expression sequence tags from haustoria of the wheat stripe rust fungus Puccinia striiformis f. sp. Tritici.

BMC Genomics 2009 Dec 23;10:626. Epub 2009 Dec 23.

Department of Plant Pathology, Washington State University, Pullman, 99164-6430, USA.

Background: Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. In spite of its agricultural importance, the genomics and genetics of the pathogen are poorly characterized. Pst transcripts from urediniospores and germinated urediniospores have been examined previously, but little is known about genes expressed during host infection. Some genes involved in virulence in other rust fungi have been found to be specifically expressed in haustoria. Therefore, the objective of this study was to generate a cDNA library to characterize genes expressed in haustoria of Pst.

Results: A total of 5,126 EST sequences of high quality were generated from haustoria of Pst, from which 287 contigs and 847 singletons were derived. Approximately 10% and 26% of the 1,134 unique sequences were homologous to proteins with known functions and hypothetical proteins, respectively. The remaining 64% of the unique sequences had no significant similarities in GenBank. Fifteen genes were predicted to be proteins secreted from Pst haustoria. Analysis of ten genes, including six secreted protein genes, using quantitative RT-PCR revealed changes in transcript levels in different developmental and infection stages of the pathogen.

Conclusions: The haustorial cDNA library was useful in identifying genes of the stripe rust fungus expressed during the infection process. From the library, we identified 15 genes encoding putative secreted proteins and six genes induced during the infection process. These genes are candidates for further studies to determine their functions in wheat-Pst interactions.
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http://dx.doi.org/10.1186/1471-2164-10-626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805700PMC
December 2009

A germin-like protein gene family functions as a complex quantitative trait locus conferring broad-spectrum disease resistance in rice.

Plant Physiol 2009 Jan 14;149(1):286-96. Epub 2008 Nov 14.

Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, Colorado 80523-1177, USA.

Plant disease resistance governed by quantitative trait loci (QTL) is predicted to be effective against a broad spectrum of pathogens and long lasting. Use of these QTL to improve crop species, however, is hindered because the genes contributing to the trait are not known. Five disease resistance QTL that colocalized with defense response genes were accumulated by marker-aided selection to develop blast-resistant varieties. One advanced backcross line carrying the major-effect QTL on chromosome (chr) 8, which included a cluster of 12 germin-like protein (OsGLP) gene members, exhibited resistance to rice (Oryza sativa) blast disease over 14 cropping seasons. To determine if OsGLP members contribute to resistance and if the resistance was broad spectrum, a highly conserved portion of the OsGLP coding region was used as an RNA interference trigger to silence a few to all expressed chr 8 OsGLP family members. Challenge with two different fungal pathogens (causal agents of rice blast and sheath blight diseases) revealed that as more chr 8 OsGLP genes were suppressed, disease susceptibility of the plants increased. Of the 12 chr 8 OsGLPs, one clustered subfamily (OsGER4) contributed most to resistance. The similarities of sequence, gene organization, and roles in disease resistance of GLP family members in rice and other cereals, including barley (Hordeum vulgare) and wheat (Triticum aestivum), suggest that resistance contributed by the chr 8 OsGLP is a broad-spectrum, basal mechanism conserved among the Gramineae. Natural selection may have preserved a whole gene family to provide a stepwise, flexible defense response to pathogen invasion.
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http://dx.doi.org/10.1104/pp.108.128348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613727PMC
January 2009

Targeted mapping of ESTs linked to the adult plant resistance gene Lr46 in wheat using synteny with rice.

Funct Integr Genomics 2006 Apr 23;6(2):122-31. Epub 2005 Dec 23.

Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA.

The gene Lr46 has provided slow-rusting resistance to leaf rust caused by Puccinia triticina in wheat (Triticum aestivum), which has remained durable for almost 30 years. Using linked markers and wheat deletion stocks, we located Lr46 in the deletion bin 1BL (0.84-0.89) comprising 5% of the 1BL arm. The distal part of chromosome 1BL of wheat is syntenic to chromosome 5L of rice. Wheat expressed sequence tags (ESTs) mapping in the terminal 15% of chromosome 1BL with significant homology to sequences from the terminal region of chromosome 5L of rice were chosen for sequence-tagged site (STS) primer design and were mapped physically and genetically. In addition, sequences from two rice bacterial artificial chromosome clones covering the targeted syntenic region were used to identify additional linked wheat ESTs. Fourteen new markers potentially linked to Lr46 were developed; eight were mapped in a segregating population. Markers flanking (2.2 cM proximal and 2.2 cM distal) and cosegregating with Lr46 were identified. The physical location of Lr46 was narrowed to a submicroscopic region between the breakpoints of deletion lines 1BL-13 [fraction length (FL)=0.89-1] and 1BL-10 (FL=0.89-3). We are now developing a high-resolution mapping population for the positional cloning of Lr46.
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http://dx.doi.org/10.1007/s10142-005-0017-9DOI Listing
April 2006

Recombination events generating a novel Rp1 race specificity.

Mol Plant Microbe Interact 2005 Mar;18(3):220-8

Department of Plant Pathology, Throckmorton Plant Science Center, Kansas State University, Manhattan, KS, USA.

Genes at the maize Rp1 rust resistance complex often mispair in meiosis, which allows genes to recombine unequally, creating recombinant haplotypes. Four recombinant haplotypes were identified from progeny of an Rp1-D/Rp1-I heterozygote that conferred a nonparental resistance specificity designated Rp1-I*. Sequence comparisons of paralogs in the recombinant and parental haplotypes demonstrated that all four recombinants were derived from intergenic (between gene) recombination events. The sequence of paralogs in the HRp1-I parental haplotype indicated this haplotype includes 41 or more rp1 genes, at least 31 of which are transcribed. The results indicate that most of the novel resistance specificities that have arisen spontaneously at Rp1 are the result of reassort ment of existing Rp1 genes.
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http://dx.doi.org/10.1094/MPMI-18-0220DOI Listing
March 2005

Identification and characterization of regions of the rice genome associated with broad-spectrum, quantitative disease resistance.

Genetics 2005 Apr 16;169(4):2277-93. Epub 2005 Feb 16.

Department of Plant Breeding and Genetics, Institute for Genomic Diversity, Cornell University, Ithaca, New York 14853, USA.

Much research has been devoted to understanding the biology of plant-pathogen interactions. The extensive genetic analysis of disease resistance in rice, coupled with the sequenced genome and genomic resources, provides the opportunity to seek convergent evidence implicating specific chromosomal segments and genes in the control of resistance. Published data on quantitative and qualitative disease resistance in rice were synthesized to evaluate the distributions of and associations among resistance loci. Quantitative trait loci (QTL) for resistance to multiple diseases and qualitative resistance loci (R genes) were clustered in the rice genome. R genes and their analogs of the nucleotide binding site-leucine-rich repeat class and genes identified on the basis of differential representation in disease-related EST libraries were significantly associated with QTL. Chromosomal segments associated with broad-spectrum quantitative disease resistance (BS-QDR) were identified. These segments contained numerous positional candidate genes identified on the basis of a range of criteria, and groups of genes belonging to two defense-associated biochemical pathways were found to underlie one BS-QDR region. Genetic dissection of disease QTL confidence intervals is needed to reduce the number of positional candidate genes for further functional analysis. This study provides a framework for future investigations of disease resistance in rice and related crop species.
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http://dx.doi.org/10.1534/genetics.104.036327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449593PMC
April 2005

Allelic and haplotypic diversity at the rp1 rust resistance locus of maize.

Genetics 2004 Aug;167(4):1939-47

Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506, USA.

The maize Rp1 rust resistance locus is a complex consisting of a family of closely related resistance genes. The number of Rp1 paralogs in different maize lines (haplotypes) varied from a single gene in some stocks of the inbred A188 to >50 genes in haplotypes carrying the Rp1-A and Rp1-H specificities. The sequences of paralogs in unrelated haplotypes differ, indicating that the genetic diversity of Rp1-related genes is extremely broad in maize. Two unrelated haplotypes with five or nine paralogs had identical resistance phenotypes (Rp1-D) encoded in genes that differed by three nucleotides resulting in a single amino acid substitution. Genes in some haplotypes are more similar to each other than to any of the genes in other haplotypes indicating that they are evolving in a concerted fashion.
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http://dx.doi.org/10.1534/genetics.104.029371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1471013PMC
August 2004

Aberrant mRNA processing of the maize Rp1-D rust resistance gene in wheat and barley.

Mol Plant Microbe Interact 2004 Aug;17(8):853-64

CSIRO Plant Industry, Box 1600, Canberra, ACT, 2601, Australia.

The maize Rp1-D gene confers race-specific resistance against Puccinia sorghi (common leaf rust) isolates containing a corresponding avrRp1-D avirulence gene. An Rp1-D genomic clone and a similar Rp1-D transgene regulated by the maize ubiquitin promoter were transformed independently into susceptible maize lines and shown to confer Rp1-D resistance, demonstrating that this resistance can be transferred as a single gene. Transfer of these functional transgenes into wheat and barley did not result in novel resistances when these plants were challenged with isolates of wheat stem rust (P. graminis), wheat leaf rust (P. triticina), or barley leaf rust (P. hordei). Regardless of the promoter employed, low levels of gene expression were observed. When constitutive promoters were used for transgene expression, a majority of Rp1-D transcripts were truncated in the nucleotide binding site-encoding region by premature polyadenylation. This aberrant mRNA processing was unrelated to gene function because an inactive version of the gene also generated such transcripts. These data demonstrate that resistance gene transfer between species may not be limited only by divergence of signaling effector molecules and pathogen avirulence ligands, but potentially also by more fundamental gene expression and transcript processing limitations.
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http://dx.doi.org/10.1094/MPMI.2004.17.8.853DOI Listing
August 2004

The avrRxo1 gene from the rice pathogen Xanthomonas oryzae pv. oryzicola confers a nonhost defense reaction on maize with resistance gene Rxo1.

Mol Plant Microbe Interact 2004 Jul;17(7):771-9

Department of Plant Pathology, 4024 Throckmorton Plant Sciences Center, Kansas State University, Manhattan 66506-5502, USA.

Maize lines that contain the single dominant gene Rxo1 exhibit a rapid hypersensitive response (HR) after infiltration with the rice bacterial streak pathogen Xanthomonas oryzae pv. oryzicola, but not with the rice bacterial blight pathogen X. oryzae pv. oryzae. The avirulence effector gene that corresponds to Rxo1, designated avrRxo1, was identified in an X. oryzae pv. oryzicola genomic library. When introduced into X. oryzae pv. oryzae, clones containing avrRxo1 induced an HR on maize with Rxo1, but not on maize without Rxo1. The avrRxo1 gene is 1,266 bp long and shows no significant homology to any database sequences. When expressed in an X. oryzae pv. oryzae hrpC mutant that is deficient in the type III secretion system, avrRxo1 did not elicit the HR, indicating that the avrRxo1-Rxo1 interaction is dependent on type III secretion. Transient expression of avrRxo1 in onion cells after biolistic delivery revealed that the protein product was associated with the plasma membrane. Transient expression in maize lines carrying Rxo1 resulted in cell death, suggesting that AvrRxo1 functions from inside maize cells to elicit Rxo1-dependent pathogen recognition.
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http://dx.doi.org/10.1094/MPMI.2004.17.7.771DOI Listing
July 2004

Diversity in nucleotide binding site-leucine-rich repeat genes in cereals.

Genome Res 2002 Dec;12(12):1871-84

Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506-5502, USA.

The diversity of the largest group of plant disease resistance genes, the nucleotide binding site-leucine-rich repeat (NBS-LRR) genes, was examined in cereals following polymerase chain reaction (PCR) cloning and database mining. NBS-LRR genes in rice are a large and diverse class with more than 600 genes, at least three to four times the complement of Arabidopsis. Most occur in small families containing one or a few cross-hybridizing members. Unlike in Arabidopsis and other dicots, the class of NBS-LRR genes coding for a Toll and mammalian interleukin-1 receptor (TIR) domain were not amplified during the evolution of the cereals. Genes coding for TIR domains are present in the rice genome, but have diverged from the NBS-LRR genes. Most cereal genes are similar in structure to the members of the non-TIR class of dicots, although many do not code for a coiled-coil domain in their amino termini. One unique class of cereal genes, with ~50 members, codes for proteins similar to the N-termini and NBS domains of resistance genes but does not code for LRR domains. The resistance gene repertoire of grasses has changed from that of dicots in their independent evolution since the two groups diverged. It is not clear whether this reflects a difference in downstream defense signaling pathways.
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http://dx.doi.org/10.1101/gr.454902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC187567PMC
December 2002

Genetic and molecular characterization of the maize rp3 rust resistance locus.

Genetics 2002 Sep;162(1):381-94

Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506-5502, USA.

In maize, the Rp3 gene confers resistance to common rust caused by Puccinia sorghi. Flanking marker analysis of rust-susceptible rp3 variants suggested that most of them arose via unequal crossing over, indicating that rp3 is a complex locus like rp1. The PIC13 probe identifies a nucleotide binding site-leucine-rich repeat (NBS-LRR) gene family that maps to the complex. Rp3 variants show losses of PIC13 family members relative to the resistant parents when probed with PIC13, indicating that the Rp3 gene is a member of this family. Gel blots and sequence analysis suggest that at least 9 family members are at the locus in most Rp3-carrying lines and that at least 5 of these are transcribed in the Rp3-A haplotype. The coding regions of 14 family members, isolated from three different Rp3-carrying haplotypes, had DNA sequence identities from 93 to 99%. Partial sequencing of clones of a BAC contig spanning the rp3 locus in the maize inbred line B73 identified five different PIC13 paralogues in a region of approximately 140 kb.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462242PMC
September 2002