11th International Cereal Rusts and Powdery Mildews Conference
John Innes
Centre,
Recommended abstract citation format:
Jafary H, Niks RE, 2004. Mapping of quantitative genes in barley determining the resistance to the heterologous wheat leaf rust fungus (Puccinia triticina). Proceedings of the 11th International Cereal Rusts and Powdery Mildews Conference, Norwich, England, 22-27 August 2004, abstract 1.4, Cereal Rusts and Powdery Mildews Bulletin [www.crpmb.org/icrpmc11/abstracts.htm].
Replace {a} by @ to use e-mail addresses
ABSTRACTS
Part 1 : Lectures
1.1 In search of durable resistance
Michele C. Heath
michele.heath{a}utoronto.ca
More than 20 years ago, Roy Johnson
coined the term “durable resistance” to describe disease resistance proven to
be effective for long periods of time. Not surprisingly, producing durable
resistance has been the goal of much plant pathological
If it is assumed that inducible defenses elicited during most examples of
nonhost resistance are the result of “pattern recognition” analogous to the
recognition of pathogen molecules (elicitors) during the innate immune response
in animals, then successful a pathogen must have adapted to “overcome” such
defenses in its host species. Understanding how a pathogen can overcome
constitutive and inducible plant defenses in its host plant is important for
developing ways of “putting back” durable resistance into these
pathosystems. The study of dikaryon infection structures suggest that initial
rust fungal development requires a sequence of plant signals as well as the
ability of the fungus to inhibit prehaustorial defenses and to prevent an HR
after haustorium formation. Studies of monokaryon infection suggests that
defenses associated with plant cell wall penetration are disrupted in the host
species by a localized reduction in adhesion between the plant cell wall and
the plasma membrane, and that protein synthesis and specific gene expression
are suppressed in a susceptible cultivar prior to the fungus entering the cell
lumen. Data suggest that although active defenses against rust fungi during
nonhost resistance are part of the same battery of defenses used by plants
against other pathogens, the mode of overcoming them may not be the same as
that used by othe
The recent rapid accumulation of information from host and nonhost plant-pathogen interactions involving biotrophic fungi means that we are rapidly reaching the point where protocols for engineering of disease resistance that is not easily overcome can be envisaged. However, the practicality of producing durable resistance in an agricultural setting still present large difficulties.
1.2 The ancient cell death suppressor BAX Inhibitor-1 induces susceptibility of barley to appropriate and inappropriate powdery mildew fungi
Ralph Hueckelhoven, Ruth Eichmann, Holger Schultheiss,
Ralph.Hueckelhoven{a}agrar.uni-giessen.de
Penetration into barley epidermal cells and subsequent haustoria formation is the key step in establishment of compatibility of barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh) and susceptible host plants. Vice versa, broad spectrum resistance, such as mediated by the recessive mutant mlo-gene, is expressed as penetration resistance with cell wall-associated defence. Interestingly, both successful penetration and apoplastic defence are accompanied by accumulation of reactive oxygen intermediates, O2•- and H2O2, respectively (1). Cell death, reactive oxygen intermediate accumulation and penetration resistance are linked in barley via regulation by the MLO protein. Therefore, we isolated the ancient cell death suppressor BAX Inhibitor-1 from barley (2, 3) and studied its role in penetration resistance to Bgh. Single-cell overexpression of wild type BAX inhibitor-1 strongly supported susceptibility of barley to Bgh (2, 3). A functional GFP:BI-1 fusion protein was detected in the ER and nuclear envelope by confocal laser scanning microscopy. Astonishingly, overexpression of BAX inhibitor-1 was sufficient to break both mlo-mediated resistance to Bgh and nonhost resistance to B. graminis f.sp. tritici (3, 4). Since BI-1 is able to suppress cell death in animals and plants as well as penetration resistance in barley, there appear to be conserved overlapping pathways that regulate cell death and defence responses, possibly in all higher eukaryotes (2).
(1) Hückelhoven and Kogel, 2003 Planta 216, 891–902
(2) Hückelhoven (in press) Apoptosis
(3) Hückelhoven et al., 2003 Proceedings of the National Academy of Science USA 100, 5555-5560
(4) Eichmann et al. (in press) Molecular Plant-Microbe Interaction
1.3 Comparison of gene expression profiles in barley epidermis in response to Blumeria graminis f.sp. hordei and Blumeria graminis f.sp. tritici
Wubei Dong, Dimitar Douchkov and Patrick Schweizer
Institute of Plant genetics and Crop Plant Research (IPK), Gatersleben, Germany
wubei.dong{a}ipk-gatersleben.de
Plants often respond in similar ways to host and nonhost pathogens (Thordal-Christensen, 2003). Up to now, most of the identified resistance mechanisms are shared by host and nonhost. Considering the clear difference between host and nonhost resistance with respect to durability, the question about the key components of nonhost resistance is appealing. We compared nonhost and host expression profiles in barley epidermis in response to Blumeria graminis f.sp. tritici (Bgt) and Blumeria graminis f.sp. hordei (Bgh) by using a barley 10k cDNA array. At 6h, 12h, 24h and 36h after inoculation, epidermis was stripped and RNA isolated. cDNA probes were labeled with 33P and hybridized to the array membranes. Clustering analysis indicated that the host and nonhost induced expression patterns are similar. Bgt and Bgh responses at earlier stages show clearer differences, mostly at 12 hours after inoculation. At later time points, the expression patterns are more similar. 276 up-regulated genes were shared by host and nonhost responses in two independent inoculation and array experiments, whereas 244 genes were nonhost specifically induced and 101 genes host specifically induced. Our results suggest that quantitative differences in the expression of pathogen regulated genes might be a critical factor in nonhost resistance. Currently we are verifying our array results by a functional screening system based on RNAi.
Thordal-Christensen H, 2003. Fresh insights into processes of nonhost resistance. Current Opinion in Plant Biology 6, 351-7.
1.4 Mapping of quantitative genes in barley determining the resistance to the heterologous wheat leaf rust fungus (Puccinia triticina)
Laboratory of Plant Breeding, Wageningen University, Wageningen, The Netherlands
Hossein.Jafary{a}wur.nl
Very little is known on the genetic
basis of non-host resistance of crops to specialised pathogens. In order to
investigate the inheritance of this resistance, we chose barley, since in this
species some accessions in the seedling stage are still somewhat susceptible to
heterologous rust species like the wheat leaf rust, Puccinia triticina,
implying that barley is nearly a non-host to this rust species. By accumulation
of genes for susceptibility we developed a barley
1.5 Suppression of various forms of rust and powdery mildew resistance of wheat and barley
Balázs Barna
Plant Protection Institute of the Hungarian Academy of Sciences, Budapest, Hungary
bbar{a}nki.hu
Heat pre-treatment of wheat seedlings significantly increased the number of pustules or lesions of stem rust on susceptible or hypersensitive resistant cultivars respectively. The reaction type did not change on either susceptible or resistant plants; however, benzimidazole pre-treatment abolished the effect of heat predisposition.
Heat shock pre-treatment of near isogenic barley lines expressing various resistance genes against powdery mildew (Blumeria graminis f. sp. hordei) decreased resistance to the pathogenic fungus.
Near isogenic lines, with and without Mla, Mlg or mlo resistance genes, of barley cvs. Pallas and Ingrid were immersed in hot water (48-49 oC) for 25 seconds one day before inoculation with Bgh race A6. Heat predisposition significantly increased the number of powdery mildew colonies on susceptible leaves, the number and size of lesions with limited sporulation on Mla lines and the number of visible lesions on Mlg lines. Heat treatment did not affect visible symptom on mlo lines.
In addition, heat pre-treatment increased susceptibility in all barley lines with or without a resistance gene by improving the development of the fungus (secondary germ tube, haustoria formation) and suppressing the resistance responses of plants (papilla formation, epidermal HR and epidermal H2O2 generation). In the mesophyll, however, elevated HR and H2O2 accumulation were observed, probably due to slight damage by heat stress.
Since, in both wheat/rust and barley /powdery mildew interactions, the susceptibility of originally susceptible plants was increased, it seems that a basal resistance, which is suppressed by heat pre-treatment, exists in all plants.
Changes in membrane leakage, antioxidants and possible heat shock proteins will be discussed in relation to the decrease in resistance.
1.6 Analysis of the mechanism of RAC/ROP GTPase activity in susceptibility of barley to the powdery mildew fungus
Holger Schultheiss, Krystina Opalski,
Institute of Phytopathology und Applied Zoology, University Giessen, Germany
holger.schultheiss{a}agrar.uni-giessen.de
Penetration of a barley epidermal cell is the crucial step in pathogenesis of the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). To prevent penetration by Bgh, attacked cells have to focus the defence machinery to the site of fungal invasion. A prerequisite for cellular polarisation is the rearrangement of the cytoskeleton. Pharmacological inhibition of cytoskeleton rearrangement leads to enhanced penetration of powdery mildew fungi into barley epidermal cells.
Small monomeric G-Proteins of the RAC/ROP family are known to be involved in the regulation of actin structure. Using a candidate RT-PCR approach we identified six cDNAs coding for small RAC/ROP GTPases in barley. The transient knock-down of HvRACB using the sequence-specific dsRNA interference technique enhanced the penetration resistance of barley to Bgh (Schultheiss et al., 2002). Vice versa, single-cell overexpression of constitutive active (CA) HvRACB reduced background resistance. Histochemical staining of actin filaments in transformed cells revealed an influence of CA RACB on the host cytoskeleton. Additionally, overexpression of CA RAC3, CA ROP4 and CA ROP6 enhanced the accessibility of barley to Bgh by an unknown mechanism. The specificity of this effect is given by the fact that CA RACD and CA RAC1 did not influence the barley - Bgh interaction (Schultheiss et al., 2003).
Analysis of GFP:HvRAC/ROP transformed cells revealed varying strengths of plasma membrane-association of barley RAC/ROPs. A close link between localisation and function of barley RAC/ROP proteins was obvious because delocalised CA RAC/ROP mutants failed to induce accessibility to Bgh.
Together, some of the RAC/ROP proteins appear to be specific barley susceptibility factors involved in processes supporting parasitic entry of Bgh into epidermal host cells.
Schultheiss H, Dechert C, Kogel K-H, Hückelhoven R, 2002. A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley. Plant Physiology 128, 1447-1454.
Schultheiss H, Dechert C, Kogel K-H, Hückelhoven R, 2003. Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus. Plant Journal 336, 589-601.
1.7 Powdery mildew, cereal cells and sustainable crop production
Tim Carver, Elena Prats, Alan Gay, Luis Mur, Barry Thomas and Hitoshi Kunoh
IGER, Aberystwyth, UK
tim.carver{a}bbsrc.ac.uk
Sustainable cereal cropping needs reliable disease control and various strategies exploiting host resistance have been proposed. Specific (R gene) resistance is unreliable because cereal mildews evolve virulence rapidly. Combining R genes within a cv. might decrease the risk, but, more simply, epidemics can be suppressed by growing cv. mixtures/multilines containing different R genes. An alternative is to breed for durable resistance. Apart from the (exceptional?) case of barley mlo resistance, durable resistance is likely to be under polygenic control and hard to manipulate. Nevertheless, independent resistance mechanisms can impede pathogenesis at different stages. Even if each mechanism has a minor effect, their combination may confer effective resistance. This conclusion arises from studies that generally use a single inoculation and precisely controlled conditions. But this is an over-simplistic approach to understanding natural complexities. For instance, in fields where leaves are challenged repeatedly, outcome of an initial attack drastically affects subsequent cellular responses through locally induced resistance or susceptibility. New evidence shows such effects can be instigated within 30 minutes of inoculation, probably being mediated via conidial extracellular material. Very recently we have also found dramatic effects of mildew attack on stomatal behaviour. In all barley lines examined, stomatal conductance was reduced during attempted fungal penetration in the light. In suscepts, stomata subsequently shut during dark periods but persistently failed to open fully in light, whereas in mlo barley normal stomatal response was largely restored by 36 hours. In contrast, where R gene resistance caused epidermal cell HR, nearby stomata locked permanently open, even in darkness, thus these apparently disease-free plants were severely compromised. Such complexities must be considered in designing strategies for exploiting resistance in sustainable production.
1.8 The role of the actin cytoskeleton in pathogen defense in barley
Marco Miklis, Uwe Zierold, Patrick Schweizer,
Max-Planck Institute for Plant Breeding, Koln, Germany
panstrug{a}mpiz-koeln.mpg.de
Barley Mlo encodes a protein with seven transmembrane domains that modulates basal defence to the powdery mildew fungus (Blumeria graminis f. sp. hordei, Bgh). Recessive mutations (mlo) in Mlo-mediate resistance to all Bgh isolates. Although calmodulin has been recently identified as MLO-interacting protein, the exact molecular mechanisms by which Mlo modulates plant defence remains to be elucidated. Re-mutagenesis of mlo-resistant plants revealed two genes (Ror1 and Ror2) that are necessary for full mlo resistance. To identify additional components, required for Mlo-mediated susceptibility or mlo resistance, we established a high-throughput approach in which we apply a combination of a single cell transient expression assay and dsRNAi-technology. Single cDNA clones of an epidermal cDNA-library of Bgh infected Hordeum vulgare leaves are introduced into a dsRNAi-vector. Both Mlo and mlo leaves were biolistically transfected with these constructs and subsequently, after pathogen challenge, investigated for an alteration of basal resistance or broad spectrum resistance, respectively.
Here we present the identification of a dsRNAi-construct compromising resistance in both genetic backgrounds (Mlo and mlo) of Hordeum vulgare leaves and further examination of the functional role of the gene putatively knocked-down.
1.9 Involvement of nitric oxide in papilla-based resistance and the hypersensitive response of barley attacked by blumeria graminis
Elena Prats, LAJ Mur and TLW Carver
Institute of Grassland and Enviromental Research, Aberystwyth,UK
elena.prats{a}bbsrc.ac.uk
Nitric oxide is emerging as a major signal in plant-pathogen interactions. In model plant-pathogen systems NO acts synergistically with reactive oxygen intermediates to orchestrate the hypersensitive response (HR). However, there is need for analyses of its role in economically important crop/pathogen interactions. The NO-specific stain, 4,5-diaminofluorescein-2-diacetate (DAF-2DA), was used to image and quantify NO production from 6 to 24 hours after the interaction of Blumeria graminis with barley (Hordeum vulgare) cv. Pallas (susceptible) and isoline P01 that carries allele Mla1 conditioning a rapid hypersensitive response (HR). Localised fluorescence after staining indicated that in both barley lines NO was abruptly generated around 10 h after inoculation (h.a.i.) beneath appressorial germ tubes at sites of papilla formation. In P01 a second burst of NO production that spread throughout attacked cells was initiated around 12 h.a.i. and this preceded whole-cell autofluorescence indicative of death due to HR. That fluorescence due to DAF-2DA staining was truly indicative of NO was supported by application of the NO scavenger, 1H-imidazol-1-yloxy-2-(4-carboxyphenyl)-4,5-dihydro-4, 4, 5, 5-tetramethyl-3-oxide (C-PTIO) which suppressed the fluorescence. In addition, C-PTIO application increased penetration frequencies in both barley lines, indicating a role for NO in papilla-based resistance. Furthermore, C-PTIO application slightly delayed cell death in P01 while, conversely, application of an NO donor, sodium nitroprusside, slightly accelerated cell death in P01 and increased cell death frequency in Pallas. The data show NO generation to be one of the earliest responses of barley epidermal cell defence against B. graminis attack and suggest its importance as a signal and regulatory factor in both the initiation and development of effective papillae, and in the HR.
1.10 Barley MLA protein abundance is controlled by RAR1 and is rate-limiting for efficient resistance to powdery mildew
Stéphane Bieri, Stefan Mauch, Qian-Hua Shen, Francesca Ceron, Ken Shirasu, Paul Schulze-Lefert
Institute of Plant Biology, University of Zürich, Switzerland
sbieri{a}botinst.unizh.ch
The complex barley Mla locus is highly polymorphic and harbors approximately 30 known race-specific resistance (R) genes to the powdery mildew fungus (Blumeria graminis f sp hordei). Nine molecularly isolated Mla powdery mildew R genes each encode CC-NB-LRR type proteins with an additional C-terminus (CT) lacking known structure/function motifs. The high sequence conservation (>90% identity) of the gene products and an identical gene structure suggests that many genetically characterized powdery mildew R genes at Mla are variants of a single gene at the complex locus. Previously, we have initiated a functional analysis by reciprocal domain swaps between MLA R proteins that confer different recognition specificities and exhibit differential requirement on RAR1/SGT1, two conserved proteins with putative co-chaperone-like functions. This enabled us to assign a role for the LRR-CT unit in recognition specificity and revealed that RAR1/SGT1 dependence can be uncoupled from recognition specificity. Transgenic barley lines expressing functional epitope-tagged MLA variants were used in biochemical experiments, indicating the existence of membrane-associated and cytoplasmic MLA pools in non-challenged plants. We have investigated MLA abundance in Rar1 wild type and rar1 mutant backgrounds. We will present evidence indicating a general role of RAR1 in controlling MLA steady state levels in non-challenged plants. Our data suggests that MLA protein abundance is a direct determinant for the efficiency of the resistance response.
KL Olesen and MF Lyngkjær
Risø National Laboratory, Roskilde, Denmark
Karen.lambaek.olesen{a}risoe.dk
Powdery mildew attack on a barley epidermal cell induces responses in both the epidermis and the underlying mesophyll tissue. Depending on the outcome of the powdery mildew attack, the responses may affect both susceptibility and resistance of the surrounding epidermal cells. However, we do not know if a pathogen attack in the mesophyll tissue from e.g. barley leaf rust, affects mildew susceptibility in the overlying epidermal cells. This was examined in the present study. Barley leaves were first inoculated with either a compatible or incompatible barley leaf rust isolate. When the rust had reached the mesophyll, the leaves were challenge inoculated with a compatible powdery mildew isolate. In both situations the epidermal cells showed reduced susceptibility towards powdery mildew, indicating that rust infection attempts in the mesophyll leads to induced resistance in the epidermis. However, the induced resistance was also observed well before the rust fungi infected the mesophyll, i.e. immediately after an epidermal cell was crossed by a rust hypha. Therefore the reduced susceptibility was more likely caused by growth of rust hyphae across the epidermal surface, than by rust attack in the mesophyll. Our finding supported this, because every time a rust hypha passed across an anticlinal cell wall, a short lateral branch was induced and below this, the epidermal cell accumulated papilla like material including autofluorescent compounds. Furthermore, the epidermal nucleus often moved towards the crossing rust hypha, indicating recognition of the hypha by the epidermal cell. To eliminate the surface effect of the rust, the abaxial side of the leaf was inoculated with rust and incubated for 5 days allowing the infection to progress all the way through the mesophyll cell layers. Then the adaxial side were challenge inoculated with mildew. This eliminated the reduced mildew susceptibility in the epidermal cells. Furthermore, epidermal cells on the adaxial leaf surface containing a rust haustorium (formed by rust growing from the abaxial surface) show increased mildew susceptibility. These epidermal cells, containing haustoria of both pathogens showed no signs of cell death and were able to support growth of both fungi at the same time. In conclusion, growth of rust hyphae across epidermal cells induces resistance to powdery mildew. Contrary, rust infection and haustorium formation in an epidermal cell (infected by rust from the inside of the leaf) strongly suppress resistance responses to powdery mildew. However, it is still unclear if responses to a pathogen in one tissue directly affect susceptibility to pathogens in other tissues in barley.
1.12 Resistance to leaf rust in wheat conferred by slow rusting gene Lr46
Julio Huerta-Espino1 and Ravi P Singh2
1INIFAP-CEVAMEX, Chapingo, Mexico. 2International Maize and Wheat Improvement Center (CIMMYT), Mexico
J.Huerta{a}cgiar.org
Leaf rust, caused by Puccinia triticina, is an important disease of wheat (Triticum aestivum) worldwide. Genes Lr34 and Lr46 are the only designated genes that confer slow rusting resistance. Chromosome substitution lines ‘Lalb(Pvn1B)’ and ‘Lalb(Parula 7D)’ carry genes Lr46 and Lr34 in chromosomes 1BL and 7DS, respectively in the susceptible background ‘Lalbahadur’. We used these lines to determine the effect of Lr46 on three components of slow rusting in adult plants in two replicated greenhouse trials. Fully extended flag leaves were uniformly inoculated with Mexican P. triticina race MCJ/SP and after incubation plants were placed in a greenhouse maintained at 15-200C. The mean latent periods of substitution lines with Lr46 and Lr34 were 117% and 126% and mean uredinium size 72% and 33% respectively, compared to that of the susceptible check. Lalbahadur, its chromosome 1B monosomic line and four Lalb(Pvn1B) substitution lines were grown in field trials for two seasons in northwestern Mexico under fungicide protected and non-protected conditions. Leaf rust epidemic was initiated by inoculating the spreader rows with race MCJ/SP. The area under the disease progress curves of susceptible Lalbahadur and its monosomic line were similar and those of substitution lines were significantly reduced to 36-43%. Losses of 48% and 46% in grain yields for the susceptible checks were significantly higher than the losses of 19-25% for the four Lr46 carrying substitution lines. Presence of Lr46 also reduced losses in grain weight, test weight, biomass, etc. From our studies we draw two conclusions: 1) both Lr46 and Lr34 have pleiotropic effects on the components of slow rusting, and 2) although Lr46 does not confer adequate protection when present alone, its presence slows down the leaf rust progress in the field and thus reduces the losses in grain yield significantly. Gene Lr46 must be combined with other slow rusting genes to achieve satisfactory control.
1.13 Modifers of disease resistance
Lesley A Boyd, James Melichar, Ruth MacCormack
John Innes Centre, Norwich, UK
lesley.boyd{a}bbsrc.ac.uk
Many mutation-derived alleles have found their way into commercial crop varieties (www-infocris.iaea.org). For cereals, the mlo allele of barley, conferring resistance to the fungal pathogen causing powdery mildew, is a well documented example (Jorgensen, 1992). The ability of a mutation-derived allele to enhance resistance would indicate either a modifier effect of the gene on the resistance process, or an alteration in a gene product that prevents the establishment of a compatible association between the plant and the pathogen. A number of mutants exhibiting enhanced resistance to the biotrophic fungal pathogens causing yellow rust, brown rust and powdery mildew have been isolated from wheat (Boyd et al., 2002). The mutant phenotypes are developmentally regulated in that resistance is expressed at specific, plant growth stages (Boyd and Minchin, 2001). Preliminary genetic analysis suggests that in many of the mutants the enhanced resistance is due to a single mutation of partial effect (Smith et al., 2004). Mapping populations are being analysed using DNA markers to identify the mutant loci responsible for the enhanced resistance to each fungal pathogen. Histological examination of yellow rust development and arrest of the pathogen in two of the mutants suggests that enhanced resistance is not due to an early, hydrogen peroxide mediated event.
Boyd LA, Minchin PN, 2001. Wheat mutants showing altered adult plant disease resistance. Euphytica 122, 361-68.
Boyd LA, Smith PH, Wilson AH, Minchin PN, 2002. Mutations in wheat showing altered field resistance to yellow and brown rust. Genome 45, 1035-40.
Jorgensen JH, 1992. Discovery, characterisation and exploitation of Mlo powdery mildew resistance in barley. Euphytica 63, 141-52.
Smith PH, Howie JA, Worland AJ, Stratford R, Boyd LA, 2004. Mutations in wheat exhibiting growth stage specific resistance to biotrophic fungal pathogens. Molecular Plant-Microbe Interactions (in press).
1.14 Flor revisited: Systems biology in barley-powdery mildew interactions
1Department of Plant Pathology and Center for Plant Responses to Environmental Stresses, 2Department of Statistics, 3Corn Insects and Crop Genetics Research, USDA-ARS, Iowa State University, Ames, USA
rpwise{a}iastate.edu
Active plant defense to microbial attack is highly dependent upon recognition events involving associated gene products in the host and the pathogen. Both perception of general and specific pathogen-associated molecules result in signal transduction cascades ultimately leading to disease resistance. General elicitors, which include proteins, glycoproteins, peptides, carbohydrates and lipids, signal the presence of the pathogen and are able to trigger defense responses in a non-cultivar specific manner. In contrast, specific effectors, encoded by pathogen avirulence genes, trigger cultivar-specific responses resulting in hyperactivation of basal defense, which is often accompanied by hypersensitive cell death. This specific recognition in plant-pathogen interactions conforms to the gene-for-gene hypothesis and is determined by direct or indirect interaction of host resistance (R) proteins and cognate pathogen-derived avirulence (AVR) effectors.
To ascertain the global framework of host gene expression during biotrophic pathogen invasion, we analyzed in parallel the mRNA abundance of 22,792 host genes throughout 36 (genotype x pathogen x time) interactions between barley (Hordeum vulgare) and Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew disease. A split-split-plot design was used to investigate near-isogenic barley lines with introgressed Mla6, Mla13, and Mla1 CC-NBS-LRR resistance alleles challenged with Bgh isolates 5874 (AvrMla6, AvrMla1) and K1 (AvrMla13, AvrMla1). A linear mixed model analysis was employed to identify genes with significant differential expression (p-value<0.0001) in incompatible and compatible barley-Bgh interactions across six time points after pathogen challenge. Twenty-two host genes, of which nine were of unknown function, exhibited highly similar patterns of up-regulation among all incompatible and compatible interactions up to 16 hours after inoculation (hai), coinciding with germination of Bgh conidiospores and formation of appressoria. In contrast, significant divergent expression was observed after 16 hai, during membrane-to-membrane contact between fungal haustoria and host epidermal cells, with notable suppression of the steady state levels of most transcripts in compatible interactions. These findings provide a link between the recognition of general and specific pathogen-associated molecules in the expression of plant defense responses and its implications on the evolution of host-specific resistance from the recognition and prevention of the pathogen’s suppression of plant basal defense.
Research supported by USDA Initiative for Future Agriculture and Food Systems (IFAFS) grant no. 2001-52100-11346, USDA National Research Initiative (NRI) grant no. 02-35300-12619, and the USDA-CSREES North American Barley Genome Project.
Torben Gjetting1, Peter Hagedorn1, Patrick Schweizer2, Michael F. Lyngkjær1
1Risø National Laboratory, Roskilde, Denmark. 2Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany
m.lyngkjaer{a}risoe.dk
Resistance or susceptibility in barley to the powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh) is determined at the single cell level. Even in ‘compatible’ interaction, attacked barley epidermal cells tries to prevent fungal penetration by reinforcing their cell wall. However, this defence is only partially efficient and a number of fungal penetration attempts will succeed and the attacked barley epidermal cell will be infected. The molecular basis of the interaction between barley and powdery mildew has been studied intensively at the leaf and tissue level. However, the mixture of infected and uninfected epidermal cells on a leaf makes it very difficult to relate Bgh-induced gene expression to resistance or susceptibility. To overcome this problem we have extracted contents from single barley epidermal cells after inoculation with Bgh, using glass micro-capillaries and micromanipulation and are for the first time able to separate induced gene expression in resistant and infected cells. Here expression data for two situations are presented: 1) the attacked barley cell resisted fungal penetration and 2) the attacked barley cell was infected by the fungus and contained a fungal haustorium. The contents of mRNA in the micro-extract was purified and processed into cDNA and amplified by PCR. The cDNA pools were used as template in gene specific PCR of selected genes, as radioactively labelled samples in dot-blot/array hybridisation (Gjetting et al., 2004). Detailed expression analysis was performed on micro-arrays spotted with 10 K barley genes and showed very different gene expression profiles for resistant and infected cells, ca. 2800 genes were significantly induced or suppressed in one of the situations. Two examples are GLP4 – encoding a germin-like protein that accumulated specifically in resistant cells, while GRP94 – encoding a molecular chaperone, accumulated in infected cells.
Gjetting T, Carver TLW, Skøtt L, Lyngkjær ML, 2004. Gene expression profiling of individual barley epidermal cells attacked by powdery mildew. Molecular Plant-Microbe Interactions 17, 729-738.
1.16 Positional cloning of powdery mildew resistance genes at the Pm3 locus of hexaploid wheat and characterization of a Pm3 haplotype
Nabila Yahiaoui, Payorm Srichumpa, Susanne Brunner and Beat Keller
Institute of Plant Biology, University of Zürich, Switzerland
nabila{a}botinst.unizh.ch
In wheat, race-specific resistance to the fungal pathogen powdery mildew (Blumeria graminis f. sp. tritici) is controlled by Pm genes. There are 10 alleles conferring resistance at the Pm3 locus (Pm3a to j) on chromosome 1AS of hexaploid bread wheat (Triticum aestivum L.). For the map-based cloning of the Pm3b gene from the large genome of hexaploid wheat, we have used a combined analysis of genomes from wheat species with different ploidy levels. Genetic mapping of Pm3b was performed in hexaploid wheat. The diploid Triticum monococcum and the tetraploid Triticum turgidum ssp. durum provided models for the A genome of hexaploid wheat and allowed us to establish a physical contig spanning the Pm3 locus. Although haplotypes at the Pm3 locus differed markedly between the three species, a large resistance gene-like gene family was consistently found at the Pm3 locus. Partial sequence conservation between resistant line Chul and T. monococcum combined with mutational analysis allowed the identification of a Pm3b candidate gene. The candidate gene, a member of the CC-NBS-LRR type of disease resistance genes conferred race-specific resistance to wheat powdery mildew in a transient single cell transformation assay. Haplotype studies in lines carrying known Pm3 specificities (Pm3a to j) indicated a good conservation of the haplotype in the Pm3 region. This led to the rapid identification of additional alleles at the Pm3 locus. The successful cloning of the Pm3 genes provides molecular tools to study disease resistance gene specificity determination and disease resistance gene evolution in cultivated and wild wheat species.
1.17 Isolation and characterization of the leaf rust resistance gene Lr10 from hexaploid wheat
Beat Keller, Catherine Feuillet, Silvia Travella, Nils Stein and Laurence Albar
University of Zurich, Switzerland
bkeller{a}botinst.unizh.ch
Subgenome map-based cloning was performed to isolate the Lr10 resistance gene on chromosome 1AS in hexaploid wheat. A T. monococcum BAC contig spanning more than 450 kb of the region orthologous to the Lr10 locus in hexaploid wheat was established and full-length sequencing of 211 kb spanning the gene identified two resistance gene analogs which cosegregate with Lr10 in more than 3000 F2 plants. The orthologs of both genes (T10rga1 and T10rga2-1A) were isolated from the resistant hexaploid wheat variety Thatcher Lr10. Haplotype studies at the Lr10 locus showed that recombination is totally suppressed between the two genes and that further genetic analysis cannot help to identify Lr10 from the two candidates. Evidence for the identity of the gene was obtained through mutational and transformation analysis: Single point mutations were identified in the T10rga1 gene in three independent EMS mutants of Thatcher Lr10. In addition, T10rga1 conferred resistance to leaf rust in transgenic wheat plants demonstrating that it is the Lr10 gene. Interestingly, leaf rust resistance was dramatically increased in transgenic plants compared to wild type. Our data demonstrate that map-based cloning is feasible in hexaploid wheat using adequate genetic, evolutionary and genomic tools.
1.18 The structure and possible origin of the barley mlo-11 mildew resistance allele
Pietro Piffanelli, Luke Ramsay,
Robbie Waugh, Abdellah Benabdelmouna, Angélique D´Hont, Karin Hinze, Jørgen
Helms Jørgensen, Paul Schulze Lefert, and Ralph Panstruga
panstrug{a}mpiz-koeln.mpg.de
Barley plants
carrying loss-of-function alleles (mlo) of the Mlo locus are
resistant against all known isolates of the widespread powdery mildew fungus.
The only mlo resistance allele recovered to date from a natural habitat,
mlo-11, was originally retrieved from Ethiopian landraces and nowadays
controls mildew resistance in the majority of cultivated European spring barley
elite varieties. Haplotype analysis revealed that the mlo-11 allele is
likely to have arisen only once after barley domestication. Resistance in mlo-11
plants is linked to a complex tandem repeat array inserted upstream of the
wild-type gene. The repeat units consist of a truncated Mlo gene
comprising 3.5 kb of 5’ regulatory plus 1.1 kb of coding sequence. The repeat
array generates aberrant transcripts that impair accumulation of Mlo
wild-type transcript as well as protein. We exploited meiotic instability of mlo-11
resistance and recovered susceptible revertants in which restoration of Mlo
function was accompanied by excision of the repeat array. We infer cis-dependent
perturbation of transcription machinery assembly by transcriptional
interference in mlo-11 plants as a likely mechanism leading to disease
resistance.
Mogens S Hovmøller and Annemarie F Justesen
Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg, Slagelse, Denmark
mogens.hovmoller{a}agrsci.dk
Genetic diversity is often low in populations of Puccinia striiformis f.sp. tritici although it may vary considerably between region, year and host variety. We have studied diversity in samples of P. striiformis from north-west Europe, north-east Africa and Central Asia, measured by AFLP variation and virulence traits assayed by extended sets of differential varieties. The sampling in NW-Europe covered the UK, France, Germany and Denmark in a period of 15 years, whereas samples from NE-Africa and Central Asia were obtained in 2002 and 2003. In all cases, they were collected from a wide range of locations and varieties in field trials and from farmers’ fields in these regions.
Diversity for AFLP was generally very low in the NW-European P. striiformis population, whereas diversity between populations from different continents was much larger. However, we also observed clonal lineages originating from very distant areas, which had almost identical AFLP patterns, suggesting that spores of the yellow rust fungus may potentially move across very large distances within a relatively short period of time. In the NW-European material, we observed some cases with diversity for virulence pathotype within clonal lineages defined by AFLP polymorphism based on up to 256 +2 primer combinations, which suggest that mutation for gain or loss of specific virulence/avirulence traits may occur fairly frequently. There was no indication for any kind of recombination within the time and area represented by the European samples.
The population dynamics of P. striiformis have a large impact on the expected control of yellow rust by host resistance, leading to either a decrease or an increase in the ability of specific sources of resistance to control yellow rust in specific areas. A striking example has recently been observed in Denmark: yellow rust was absent under natural conditions in 1996, indicating an extinction of the P. striiformis population in Denmark at that time; the subsequent re-colonization of a new population from external sources resulted in a complete change in susceptibility for many varieties. For instance, some of the most widely grown varieties between 1997 and 2003 were fully resistant under field conditions, although the underlying source of resistance was largely ineffective for yellow rust control in the early 1990s. This points to the need for ongoing monitoring of virulence dynamics in different wheat growing regions and for encouragement of breeders to select sources of resistance which are less vulnerable to the inevitable and continual dispersal and evolution of P. striiformis.
1.20 Clonality of wheat yellow rust population in France and high diversity in China
Jérôme Enjalbert, X Duan, A Wan, M Leconte, C de Vallavieille-Pope
INRA Grignon, France
enjalber{a}grignon.inra.fr
Puccinia striiformis f.sp. tritici (PST) has long story of breaking down cultivar resistance genes used by breeders. In Europe, a clonal behaviour of the species has been demonstrated using molecular markers, associated with a long distance dispersion capacity. Despite these spore flows, French PST populations present a strong geographic structure for virulence genes, the northern and southern population possessing different pathotypes. Using microsatellite and AFLP markers we described the neutral diversity underlying this differentiation on highly selected genes by analysing the genetic structure of 356 French isolates belonging to 10 pathotypes collected over a 12-year period. Both on microsatellite and AFLP polymorphisms, specific alleles were described for the South-specific pathotype. This strong genetic distance reveals that northern French population belongs to the North-Western European population, whereas the southern population is related to a more extended Mediterranean population, the two sub-populations resulting from the divergence of two clonal lineages. Such steady polymorphism should be explained on the basis local adaptation of the two clonal sub-populations.
Using the s