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1.  A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots 
PLoS ONE  2011;6(10):e26468.
Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.
doi:10.1371/journal.pone.0026468
PMCID: PMC3198768  PMID: 22031834
2.  Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families 
Journal of Virology  2002;76(24):12981-12991.
RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the γb gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the γb protein may be a long-distance movement factor and have antisilencing activity. This was shown for γb proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, γb and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the γb cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus γb proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.
doi:10.1128/JVI.76.24.12981-12991.2002
PMCID: PMC136670  PMID: 12438624
3.  Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat 
Plant Methods  2010;6:26.
Background
Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.
Results
Here we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-β-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS) gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa) and diploid (A. strigosa) oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component.
Conclusions
Our results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments.
doi:10.1186/1746-4811-6-26
PMCID: PMC3006357  PMID: 21118486
4.  Interactions of the TGB1 Protein during Cell-to-Cell Movement of Barley Stripe Mosaic Virus 
Journal of Virology  2001;75(18):8712-8723.
We have recently used a green fluorescent protein (GFP) fusion to the γb protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The γb protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAβ but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, βb (TGB1), βc (TGB3), and βd (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65–75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.
doi:10.1128/JVI.75.18.8712-8723.2001
PMCID: PMC115116  PMID: 11507216
5.  A complete ancient RNA genome: identification, reconstruction and evolutionary history of archaeological Barley Stripe Mosaic Virus 
Scientific Reports  2014;4:4003.
The origins of many plant diseases appear to be recent and associated with the rise of domestication, the spread of agriculture or recent global movements of crops. Distinguishing between these possibilities is problematic because of the difficulty of determining rates of molecular evolution over short time frames. Heterochronous approaches using recent and historical samples show that plant viruses exhibit highly variable and often rapid rates of molecular evolution. The accuracy of estimated evolution rates and age of origin can be greatly improved with the inclusion of older molecular data from archaeological material. Here we present the first reconstruction of an archaeological RNA genome, which is of Barley Stripe Mosaic Virus (BSMV) isolated from barley grain ~750 years of age. Phylogenetic analysis of BSMV that includes this genome indicates the divergence of BSMV and its closest relative prior to this time, most likely around 2000 years ago. However, exclusion of the archaeological data results in an apparently much more recent origin of the virus that postdates even the archaeological sample. We conclude that this viral lineage originated in the Near East or North Africa, and spread to North America and East Asia with their hosts along historical trade routes.
doi:10.1038/srep04003
PMCID: PMC3915304  PMID: 24499968
6.  Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance 
Journal of Experimental Botany  2013;64(5):1381-1392.
In a non-model staple crop like wheat (Triticum aestivumI L.), functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for breeding. Virus-induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited transformation potential that hamper functional validation studies in wheat. In this study, three potential candidate genes shown to be involved in abiotic stress response pathways in Arabidopsis thaliana were selected for VIGS experiments in wheat. These include Era1 (enhanced response to abscisic acid), Cyp707a (ABA 8’-hydroxylase), and Sal1 (inositol polyphosphate 1-phosphatase). Gene homologues for these three genes were identified in wheat and cloned in the viral vector barley stripe mosaic virus (BSMV) in the antisense direction, followed by rub inoculation of BSMV viral RNA transcripts onto wheat plants. Quantitative real-time PCR showed that VIGS-treated wheat plants had significant reductions in target gene transcripts. When VIGS-treated plants generated for Era1 and Sal1 were subjected to limiting water conditions, they showed increased relative water content, improved water use efficiency, reduced gas exchange, and better vigour compared to water-stressed control plants inoculated with RNA from the empty viral vector (BSMV0). In comparison, the Cyp707a-silenced plants showed no improvement over BSMV0-inoculated plants under limited water condition. These results indicate that Era1 and Sal1 play important roles in conferring drought tolerance in wheat. Other traits affected by Era1 silencing were also studied. Delayed seed germination in Era1-silenced plants suggests this gene may be a useful target for developing resistance to pre-harvest sprouting.
doi:10.1093/jxb/ert003
PMCID: PMC3598424  PMID: 23364940
Arabidopsis; drought; Era1; Sal1; virus-induced gene silencing; wheat.
7.  Structural Lability of Barley Stripe Mosaic Virus Virions 
PLoS ONE  2013;8(4):e60942.
Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed.
doi:10.1371/journal.pone.0060942
PMCID: PMC3629216  PMID: 23613760
8.  The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein. 
Journal of Virology  1997;71(2):1538-1546.
The barley stripe mosaic virus (BSMV) beta(b) gene product is the major viral nonstructural protein synthesized during early stages of the infection cycle and is required for systemic movement of the virus. To examine the biochemical properties of beta(b), a histidine tag was engineered at the amino terminus and the protein was purified from BSMV-infected barley tissue by metal affinity chromatography. The beta(b) protein bound ATPs in vitro, with a preference for ATP over dATP, and also exhibited ATPase activity. In addition, beta(b) bound RNA without detectable sequence specificity. However, binding was selective, as the beta(b) protein had a strong affinity for both single-stranded (ss) and double-stranded (ds) RNAs but not for tRNA or DNA substrates. Mutational analyses of beta(b) purified from Escherichia coli indicated that the protein has multiple RNA binding sites. These sites appear to contribute differently, because mutants that were altered in their binding affinities for ss and ds RNA substrates were recovered.
PMCID: PMC191211  PMID: 8995680
9.  Triple Gene Block Protein Interactions Involved in Movement of Barley Stripe Mosaic Virus▿  
Journal of Virology  2008;82(10):4991-5006.
Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.
doi:10.1128/JVI.02586-07
PMCID: PMC2346763  PMID: 18353960
10.  Nucleotide sequence of the 3'-terminal tRNA-like structure in barley stripe mosaic virus genome. 
Nucleic Acids Research  1984;12(9):4001-4009.
This paper describes the sequence of 257 nucleotides from the 3' end of RNA 2 of barley stripe mosaic virus ( BSMV , strain Argentina Mild) including an internal oligo (A) tract localized at a distance of 236 nucleotides from the 3' end, and the 3' terminal tRNA-like structure accepting tyrosine. This sequence is shown to be the same with RNAs 1,2 and 3 of another BSMV strain, Norwich , for at least the first 106 nucleotides from the 3' end. The 3' extremity of BSMV RNA bears some resemblance to tRNATyr from yeast in its primary structure. The possible secondary structures of the tRNA-like sequence in BSMV genome are discussed.
PMCID: PMC318806  PMID: 6328450
11.  Functional Characterization of Calcineurin Homologs PsCNA1/PsCNB1 in Puccinia striiformis f. sp. tritici Using a Host-Induced RNAi System 
PLoS ONE  2012;7(11):e49262.
Calcineurin plays a key role in morphogenesis, pathogenesis and drug resistance in most fungi. However, the function of calcineurin genes in Puccinia striiformis f. sp. tritici (Pst) is unclear. We identified and characterized the calcineurin genes PsCNA1 and PsCNB1 in Pst. Phylogenetic analyses indicate that PsCNA1 and PsCNB1 form a calcium/calmodulin regulated protein phosphatase belonging to the calcineurin heterodimers composed of subunits A and B. Quantitative RT-PCR analyses revealed that both PsCNA1 and PsCNB1 expression reached their maximum in the stage of haustorium formation, which is one day after inoculation. Using barely stripe mosaic virus (BSMV) as a transient expression vector in wheat, the expression of PsCNA1 and PsCNB1 in Pst was suppressed, leading to slower extension of fungal hyphae and reduced production of urediospores. The immune-suppressive drugs cyclosporin A and FK506 markedly reduced the germination rates of urediospores, and when germination did occur, more than two germtubes were produced. These results suggest that the calcineurin signaling pathway participates in stripe rust morphogenetic differentiation, especially the formation of haustoria during the early stage of infection and during the production of urediospores. Therefore PsCNA1 and PsCNB1 can be considered important pathogenicity genes involved in the wheat-Pst interaction.
doi:10.1371/journal.pone.0049262
PMCID: PMC3490909  PMID: 23139840
12.  Fine Mapping of the Bsr1 Barley Stripe Mosaic Virus Resistance Gene in the Model Grass Brachypodium distachyon 
PLoS ONE  2012;7(6):e38333.
The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25°C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F6∶7 recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F2 population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.
doi:10.1371/journal.pone.0038333
PMCID: PMC3366947  PMID: 22675544
13.  The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus. 
Nucleic Acids Research  1986;14(9):3895-3909.
The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.
Images
PMCID: PMC339823  PMID: 3754962
14.  Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins▿  
Journal of Virology  2009;83(18):9432-9448.
Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.
doi:10.1128/JVI.00739-09
PMCID: PMC2738231  PMID: 19570874
15.  Mixed model association scans of multi-environmental trial data reveal major loci controlling yield and yield related traits in Hordeum vulgare in Mediterranean environments 
An association panel consisting of 185 accessions representative of the barley germplasm cultivated in the Mediterranean basin was used to localise quantitative trait loci (QTL) controlling grain yield and yield related traits. The germplasm set was genotyped with 1,536 SNP markers and tested for associations with phenotypic data gathered over 2 years for a total of 24 year × location combinations under a broad range of environmental conditions. Analysis of multi-environmental trial (MET) data by fitting a mixed model with kinship estimates detected from two to seven QTL for the major components of yield including 1000 kernel weight, grains per spike and spikes per m2, as well as heading date, harvest index and plant height. Several of the associations involved SNPs tightly linked to known major genes determining spike morphology in barley (vrs1 and int-c). Similarly, the largest QTL for heading date co-locates with SNPs linked with eam6, a major locus for heading date in barley for autumn sown conditions. Co-localization of several QTL related to yield components traits suggest that major developmental loci may be linked to most of the associations. This study highlights the potential of association genetics to identify genetic variants controlling complex traits.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-011-1537-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-011-1537-4
PMCID: PMC3075395  PMID: 21279625
16.  Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat 
PLoS ONE  2013;8(12):e82079.
Background
Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1.
Methods
The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1+NIL were also compared to identify pathogen-responsive proteins.
Results
Eight proteins were either induced or upregulated in inoculated Fhb1+NIL when compared with mock-inoculated Fhb1+NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1+NIL when compared with Fusarium-inoculated Fhb1−NIL. Proteins that were differentially expressed in the Fhb1+NIL, not in the Fhb1−NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification.
Conclusions
Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1+NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance.
doi:10.1371/journal.pone.0082079
PMCID: PMC3869672  PMID: 24376514
17.  Quantitative and Qualitative Stem Rust Resistance Factors in Barley Are Associated with Transcriptional Suppression of Defense Regulons 
PLoS Genetics  2011;7(7):e1002208.
Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement of R-gene mediated resistance.
Author Summary
An important step in molecular plant pathology is the identification of the biologically relevant events that are directly involved in mediating resistance to pathogens. Historically, it is known that de novo and modulated gene expression are important components of the immune response. And yet, how exactly regulatory cascades orchestrate transcriptional responses to influence immunity remains unexplored. Several molecular tools have enabled the dissection of the defense transcriptome. One such technique, expression Quantitative Trait Locus (eQTL) analysis, provides the opportunity to identify genes involved in transcriptional regulation and simultaneously identifying their downstream targets. This paper describes an eQTL analysis of a barley population segregating for qualitative and quantitative immunity to stem rust (Puccinia graminis f. sp. tritici), a devastating pathogen of Triticeae grain crops. Analysis of treatment-specific effects identified several regulatory loci that alter the expression of many inoculation responsive genes. On chromosome 2H, a trans-eQTL hotspot coincides with an enhancer of adult plant resistance. Notably, the resistance allele for this locus is associated with suppressing the transcription for hundreds of genes, with some of these having been previously associated with plant disease defense. In this respect, conventional wisdom is challenged by these findings.
doi:10.1371/journal.pgen.1002208
PMCID: PMC3145622  PMID: 21829384
18.  Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum 
Journal of Experimental Botany  2008;59(9):2371-2378.
Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions.
doi:10.1093/jxb/ern103
PMCID: PMC2423652  PMID: 18467324
Chitinase; Fusarium graminearum; Fusarium head blight; transformation; wheat
19.  Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum 
BMC Plant Biology  2012;12:33.
Background
The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by Fusarium graminearum Schwabe, a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) that reduces both grain yield and quality.
Results
A construct containing a bovine lactoferrin cDNA was used to transform wheat using an Agrobacterium-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus F. graminearum. Transgenic wheat showed a significant reduction of disease incidence caused by F. graminearum compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance.
Conclusions
Introgression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to F. graminearum.
doi:10.1186/1471-2229-12-33
PMCID: PMC3364854  PMID: 22405032
20.  Identification of expressed genes during compatible interaction between stripe rust (Puccinia striiformis) and wheat using a cDNA library 
BMC Genomics  2009;10:586.
Background
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. To establish compatibility with the host, Pst forms special infection structures to invade the plant with minimal damage to host cells. Although compatible interaction between wheat and Pst has been studied using various approaches, research on molecular mechanisms of the interaction is limited. The aim of this study was to develop an EST database of wheat infected by Pst in order to determine transcription profiles of genes involved in compatible wheat-Pst interaction.
Results
Total RNA, extracted from susceptible infected wheat leaves harvested at 3, 5 and 8 days post inoculation (dpi), was used to create a cDNA library, from which 5,793 ESTs with high quality were obtained and clustered into 583 contigs and 2,160 singletons to give a set of 2,743 unisequences (GenBank accessions: GR302385 to GR305127). The BLASTx program was used to search for homologous genes of the unisequences in the GenBank non-redundant protein database. Of the 2,743 unisequences, 52.8% (the largest category) were highly homologous to plant genes; 16.3% to fungal genes and 30% of no-hit. The functional classification of all ESTs was established based on the database entry giving the best E-value using the Bevan's classification categories. About 50% of the ESTs were significantly homologous to genes encoding proteins with known functions; 20% were similar to genes encoding proteins with unknown functions and 30% did not have significant homology to any sequence in the database. The quantitative real-time PCR (qRT-PCR) analysis determined the transcription profiles and their involvement in the wheat-Pst interaction for seven of the gene.
Conclusion
The cDNA library is useful for identifying the functional genes involved in the wheat-Pst compatible interaction, and established a new database for studying Pst pathogenesis genes and wheat defense genes. The transcription patterns of seven genes were confirmed by the qRT-PCR assay to be differentially expressed in wheat-Pst compatible and incompatible interaction.
doi:10.1186/1471-2164-10-586
PMCID: PMC3087560  PMID: 19995415
21.  A jacalin-related lectin-like gene in wheat is a component of the plant defence system 
Journal of Experimental Botany  2011;62(15):5471-5483.
Jacalin-related lectins (JRLs) are a subgroup of proteins with one or more jacalin-like lectin domains. Although JRLs are often associated with biotic or abiotic stimuli, their biological functions in plants, as well as their relationships to plant disease resistance, are poorly understood. A mannose-specific JRL (mJRL)-like gene (TaJRLL1) that is mainly expressed in stem and spike and encodes a protein with two jacalin-like lectin domains was identified in wheat. Pathogen infection and phytohormone treatments induced its expression; while application of the salicylic acid (SA) biosynthesis inhibitor paclobutrazol and the jasmonic acid (JA) biosynthesis inhibitor diethyldithiocarbamic acid, respectively, substantially inhibited its expression. Attenuating TaJRLL1 through virus-induced gene silencing increased susceptibility to the facultative fungal pathogen Fusarium graminearum and the biotrophic fungal pathogen Blumeria graminis. Arabidopsis thaliana transformed with TaJRLL1 displayed increased resistance to F. graminearum and Botrytis cinerea. JA and SA levels in transgenic Arabidopsis increased significantly. A loss or increase of disease resistance due to an alteration in TaJRLL1 function was correlated with attenuation or enhancement of the SA- and JA-dependent defence signalling pathways. These results suggest that TaJRLL1 could be a component of the SA- and JA-dependent defence signalling pathways.
doi:10.1093/jxb/err226
PMCID: PMC3223046  PMID: 21862481
Defence signalling; disease resistance; jacalin-related lectin; jasmonic acid; salicylic acid; wheat (Triticum aestivum L.)
22.  Survival of the biocontrol agents Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116 on the spikes of barley in the field*  
Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease that results in extensive yield losses to wheat and barley. A green fluorescent protein (GFP) expressing plasmid pRP22-GFP was constructed for monitoring the colonization of two biocontrol agents, Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116, on the spikes of barley and their effect on suppression of FHB. Survival and colonization of the Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116 strains on spikes of barley were observed by tracking the bacterial transformants with GFP expression. Our field study revealed that plasmid pRP22-GFP was stably maintained in the bacterial strains without selective pressure. The retrieved GFP-tagged strains showed that the bacterial population fluctuation accorded with that of the rain events. Furthermore, both biocontrol strains gave significant protection against FHB on spikes of barley in fields. The greater suppression of barley FHB disease was resulted from the treatment of barley spikes with biocontrol agents before inoculation with F. graminearum.
doi:10.1631/jzus.2005.B0770
PMCID: PMC1389858  PMID: 16052710
GFP; Survival; Brevibacillus brevis and Bacillus subtilis; Spikes; Barley; Biocontrol
23.  Simultaneous detection of Fusarium culmorum and F. graminearum in plant material by duplex PCR with melting curve analysis 
BMC Microbiology  2006;6:4.
Background
Fusarium head blight (FHB) is a disease of cereal crops, which has a severe impact on wheat and barley production worldwide. Apart from reducing the yield and impairing grain quality, FHB leads to contamination of grain with toxic secondary metabolites (mycotoxins), which pose a health risk to humans and livestock. The Fusarium species primarily involved in FHB are F. graminearum and F. culmorum. A key prerequisite for a reduction in the incidence of FHB is an understanding of its epidemiology.
Results
We describe a duplex-PCR-based method for the simultaneous detection of F. culmorum and F. graminearum in plant material. Species-specific PCR products are identified by melting curve analysis performed in a real-time thermocycler in the presence of the fluorescent dye SYBR Green I. In contrast to multiplex real-time PCR assays, the method does not use doubly labeled hybridization probes.
Conclusion
PCR with product differentiation by melting curve analysis offers a cost-effective means of qualitative analysis for the presence of F. culmorum and F. graminearum in plant material. This method is particularly suitable for epidemiological studies involving a large number of samples.
doi:10.1186/1471-2180-6-4
PMCID: PMC1388219  PMID: 16430784
24.  Genetic Insight into Yield-Associated Traits of Wheat Grown in Multiple Rain-Fed Environments 
PLoS ONE  2012;7(2):e31249.
Background
Grain yield is a key economic driver of successful wheat production. Due to its complex nature, little is known regarding its genetic control. The goal of this study was to identify important quantitative trait loci (QTL) directly and indirectly affecting grain yield using doubled haploid lines derived from a cross between Hanxuan 10 and Lumai 14.
Methodology/Principal Findings
Ten yield-associated traits, including yield per plant (YP), number of spikes per plant (NSP), number of grains per spike (NGS), one-thousand grain weight (TGW), total number of spikelets per spike (TNSS), number of sterile spikelets per spike (NSSS), proportion of fertile spikelets per spike (PFSS), spike length (SL), density of spikelets per spike (DSS) and plant height (PH), were assessed across 14 (for YP) to 23 (for TGW) year × location × water regime environments in China. Then, the genetic effects were partitioned into additive main effects (a), epistatic main effects (aa) and their environment interaction effects (ae and aae) by using composite interval mapping in a mixed linear model.
Conclusions/Significance
Twelve (YP) to 33 (PH) QTLs were identified on all 21 chromosomes except 6D. QTLs were more frequently observed on chromosomes 1B, 2B, 2D, 5A and 6B, and were concentrated in a few regions on individual chromosomes, exemplified by three striking yield-related QTL clusters on chromosomes 2B, 1B and 4B that explained the correlations between YP and other traits. The additive main-effect QTLs contributed more phenotypic variation than the epistasis and environmental interaction. Consistent with agronomic analyses, a group of progeny derived by selecting TGW and NGS, with higher grain yield, had an increased frequency of QTL for high YP, NGS, TGW, TNSS, PFSS, SL, PH and fewer NSSS, when compared to low yielding progeny. This indicated that it is feasible by marker-assisted selection to facilitate wheat production.
doi:10.1371/journal.pone.0031249
PMCID: PMC3281929  PMID: 22363596
25.  The Effect of High Concentrations of Glufosinate Ammonium on the Yield Components of Transgenic Spring Wheat (Triticum aestivum L.) Constitutively Expressing the bar Gene 
The Scientific World Journal  2012;2012:657945.
We present an experiment done on a bar+ wheat line treated with 14 different concentrations of glufosinate ammonium—an effective component of nonselective herbicides—during seed germination in a closed experimental system. Yield components as number of spikes per plant, number of grains per spike, thousand kernel weight, and yield per plant were thoroughly analysed and statistically evaluated after harvesting. We found that a concentration of glufosinate ammonium 5000 times the lethal dose was not enough to inhibit the germination of transgenic plants expressing the bar gene. Extremely high concentrations of glufosinate ammonium caused a bushy phenotype, significantly lower numbers of grains per spike, and thousand kernel weights. Concerning the productivity, we observed that concentrations of glufosinate ammonium 64 times the lethal dose did not lead to yield depression. Our results draw attention to the possibilities implied in the transgenic approaches.
doi:10.1100/2012/657945
PMCID: PMC3354593  PMID: 22649303

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