Basal Resistance of Barley to Adapted and Non-Adapted Forms of Blumeria Graminis

Home , Blumeria graminis, Erysiphales, Erysiphe, Oidium (genus)

Basal resistance of barley to adapted and non-adapted forms of Blumeria graminis

Reza Aghnoum

Thesis committee

Thesis supervisors Prof. Dr. Richard G.F. Visser Professor of Plant Breeding Wageningen University

Dr.ir. Rients E. Niks Assistant professor, Laboratory of Plant Breeding Wageningen University

Other members Prof. Dr. R.F. Hoekstra, Wageningen University Prof. Dr. F. Govers, Wageningen University Prof. Dr. ir. C. Pieterse, Utrecht University Dr.ir. G.H.J. Kema, Plant Research International, Wageningen

This research was conducted under the auspices of the Graduate school of Experimental Plant Sciences.

Basal resistance of barley to adapted and non-adapted forms of Blumeria graminis

Reza Aghnoum

Thesis Submitted in partial fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Doctorate Board to be defended in public on Tuesday 16 June 2009 at 4 PM in the Aula.

III

Reza Aghnoum Basal resistance of barley to adapted and non-adapted forms of Blumeria graminis 132 pages.

Thesis, Wageningen University, Wageningen, NL (2009) With references, with summaries in Dutch and English

ISBN 978-90-8585-419-7

IV Contents

Chapter 1 1 General introduction Chapter 2 15 Which candidate genes are responsible for natural variation in basal resistance of barley to barley powdery mildew? Chapter 3 47 Transgressive segregation for extreme low and high level of basal resistance to powdery mildew in barley Chapter 4 61 New perspectives to identify genes that determine non-host resistance of barley to non-adapted Blumeria graminis forms Chapter 5 79 Rust-induced powdery mildew resistance in barley Chapter 6 101 General discussion Abbreviations 111

Glossary 113

Summary in English 115

Summary in Dutch (Samenvatting) 119

Acknowledgments 123

About the author 127

List of publications 129

Education certificate 131

This thesis is dedicated with deep gratitude to my dear wife Fatemeh and our lovely children, Mahrokh and Pouria

General Introduction

Chapter 1

Introduction Powdery mildews, a large group of Ascomycota fungal species, are obligate biotrophs that colonize a wide range of plant species. These fungi consist of 16 genera and about 650 species (Inuma et al ., 2007). Nearly 10,000 species of angiosperms have been reported as host plants for powdery mildews (Braun, 2002). The host range of these fungi is restricted to angiosperms, and does not include ferns and gymnosperms (Takamatsu, 2004). They cause economic loss on a wide range of important agricultural crops, including cereals. Powdery mildew of the grass family is caused exclusively by members of the species Blumeria graminis. On the basis of host range, B. graminis is divided into formae speciales (f.sp., plural ff.spp.), each of which can infect mainly species of one genus of gramineous plants (Wyand & Brown, 2003). Powdery mildew of barley caused by Blumeria gramins (DC.) Speer f.sp. hordei Em. Marchal (syn. Erysiphe graminis DC. f.sp. hordei Em. Marchal) is one of the most important diseases of barley in Europe and many major barley cultivation areas.

Barley-Blumeria interaction as a model system Due to several reasons, powdery mildew of barley has been studied extensively and it can be considered as an experimental model system for the study of host-pathogen interactions. 1- The development of the fungus is restricted to the host epidermal surface, so the successive developmental stages of germlings including germination, appressorial germ tube growth and haustorium formation can be easily monitored by light microscopy. The synchronized development of the fungus on the host facilitates meaningful experiments where physiological and molecular responses of the barley host can be correlated perfectly with the development of the fungus, using bioimaging analyses and transcript profiling (Collinge et al. , 2008). 2- The mode of infection by mildew exclusively involves the epidermis. Epidermal cells can be transiently transformed relatively easily and transient-induced gene silencing (TIGS) and transient over-expression in single epidermal cells can be applied, allowing reverse genetics to identify genes that influence the barley-Blumeria interaction (Douchkov et al ., 2005). 3-There is a great availability of valuable genetic stock. More than 85 race-specific resistance genes for powdery mildew have been identified in barley (Jørgensen, 1994). Many near-isogenic lines (see Glossary) have been developed by back- crossing different resistance gene donors into common genetic background, resulting in, e.g., the Pallas differential set (Kølster et al ., 1984). Such sets of near-isogenic

2 General introduction

lines form excellent experimental material for comparative studies of defense responses (Thordal-Christensen et al ., 2000). The incompatible phenotype of different R genes/barley mildew interactions varies from single cell to multi-cell hypersensitive reaction (HR) or are based on formation of papillae with a subsequent HR. So far six major resistance genes of barley to powdery mildew ( Mla1 , Mla6, Mla7, Mla10, Mla12 and Mla13 ) have been cloned and characterized (Chełkowski et al. , 2003, Stein & Graner, 2004). Furthermore, some genes have been cloned that are required for R- gene-mediated resistance viz. Rar1 (required for Mla12 resistance-1) and Sgt1 (suppressor of the G2 allele of skp1 ) (Kitagawa et al. , 1999; Azevedo et al ., 2002). 4- Barley is the first crop in which an alternative, durable (see Glossary) type of resistance, mlo resistance, has been found. This resistance is mediated by the recessive mlo allele of the Mlo (Mildew resistance locus-o) locus. mlo resistance is effective against all known isolates of the barley powdery mildew fungus (Jørgensen, 1992;Büschges et al ., 1997), and has remained so despite extensive cultivation of mlo -cultivars in Europe since 1979 (Lyngkjær et al ., 2000). mlo resistance is associated with formation of cell wall appositions (papillae) at the site of attempted fungal penetration. Cloning of the Mlo wild type allele and Ror2 , a gene that is required for mlo-mediated resistance, provided insights into papilla-based penetration resistance (Büschges et al ., 1997; Collins et al ., 2003). Non-host resistance of barley against non-adapted powdery mildews, the basal host resistance and mlo -mediated resistance are based on similar mechanisms (Trujillo et al ., 2004; Humphry et al ., 2006; Schweizer, 2007). Only recently, more than 60 years after the first report of mlo in barley, effective homologues have been found in tomato, pea and Arabidopsis thaliana (Collins et al ., 2003; Fondevilla et al ., 2006; Bai et al ., 2008). In Arabidopsis thaliana these genes are called penetration mutant genes ( PEN ), and they have been shown to be required for both non-host resistance and a host resistance that is similar to the ml o resistance in barley to B. graminis (Consonni et al ., 2006). 5. Barley is a diploid autogamous crop, in which DH production is technically feasible. This is an advantage over an allogamous cereal crop like rye (heterozygous) and allopolyploid crops like wheat and oat that possess in principle two or three homologous copies of gene of interest, one on each genome (Elliott et al ., 2002).

Barley-powdery mildew: Compatible interaction The powdery mildew pathogen disperses by asexually formed windborne conidia. Approximately 2 hours after contact with a leaf surface, the conidium germinates with a primary germ tube (PGT) (Figure 1). This germ tube penetrates through the leaf cuticle and may have a function in conidial adhesion, water uptake, recognition of leaf

3 Chapter 1

surface structures and perception and transduction of leaf derived signals (Heitefuss, 2001; Eichmann & Hückelhoven, 2008). Within about 4 h, a secondary germ tube, that is called the appressorial germ tube (AGT), will be formed. AGT differentiates an appressorium by 8-12 h, from which the fungus tries to penetrate the host cell wall by a penetration peg. Attacked epidermal cells react to the fungal penetration peg by papilla deposition. If fungal penetration succeeds, a haustorium will be formed in the attacked cell. This is an organ by which the fungus obtains its nutrition from the plant cells and probably organizes reprogramming of the plant cell’s gene expression. After successful formation of the first haustorium (called establishment) an elongated secondary hypha (ESH) will start to grow from the primary appressorium. From these ESH, secondary appressoria are formed that will penetrate epidermal cells to establish secondary haustoria. Three to four days after formation of the first haustorium, the pathogen develops conidiophores, on which chains of conidia are formed. Therefore this development stage is also called conidiation. Released mature conidia can initiate a new infection cycle (Figure 1).

Penetration resistance in barley-powdery mildew interaction Penetration thorough the epidermal cell walls is a critical stage during the infection process of powdery mildews. Epidermal cells actively resist penetration by powdery mildews in association with papilla formation at the site of attempted fungal penetration, and hence may prevent haustorium formation (Figure 1). Papillae consist of a variety of compounds, including callose, phenolic compounds, H 2O2, proteins and glycoproteins with hydrolytic properties (Zeyen et al ., 2002). Even in a susceptible plant some germlings fail to penetrate in association with papilla formation. The speed of papilla deposition and their chemical composition play an important role in the success or failure of haustorium formation at that site (Heitefuss, 2001). Upon failure of attempted penetration from the first appressorial lobe, the appressorium may form one or two subsequent lobes to try again (Carver, 1986). In interaction of barley with the powdery mildew pathogen, three types of resistance, viz. basal host resistance, non-host resistance and mlo -mediated resistance operate on the basis of similar mechanisms, viz. penetration resistance in association with papilla formation.

Basal host resistance Even in susceptible plants a proportion of germlings stop at penetration stage in association with papilla formation due to basal resistance. This resistance is defined as the plant’s ability to reduce the disease severity caused by an adapted pathogen

4 General introduction

despite a compatible interaction phenotype. Histological studies on apparently susceptible barley accessions showed that resistance to primary penetration varies greatly (e.g. Carver, 1986; Asher & Thomas, 1987). Penetration resistance to powdery mildew in barley also depends on the type of the epidermal cells. Higher frequencies of successful penetration occurs on the short cells which directly are in contact with the stomata (epidermal cells type A) and short cells not directly adjacent

72hai ESH 5-8 dai C AGT 12hai PA PGT

::: :::

Papillae defense Hypersensitive response Compatible interaction mlo -mediated, Non-host R genes, Mla1 , Mla6 etc . HAU and basal host resistance

Figure 1. Schematic representation of different outcome of interaction of barley with the powdery mildew pathogen. hai/dai = hours/days after inoculation, PGT=Primary germ tube AGT=Appressorial germ tube, C=Conidia, HAU=Haustorium, ESH= Elongated secondary hyphae, Pa=Papilla to stomata (type B) than on the long epidermal cells covering the vascular tissue (Lin & Edwards, 1974; Koga et al ., 1990). Infection rate in both short and long epidermal cells also depends on the leaf age. On older leaves, a lower proportion of germlings succeed to establish than on younger leaves (Lin & Edwards, 1974; Carver, 1986). Basal resistance is a quantitatively inherited trait, conferred by many, mostly additively acting genes, each with small effect, so it is also called quantitative resistance. Advances in molecular marker technology and QTL analysis have led to identification of many QTLs in different genetic backgrounds of barley, but the genes underlying the resistance QTLs are still elusive. Using transient-induced gene silencing and transient over-expression in single epidermal cells, many candidate genes have been identified that play a role in host, non-host and/or mlo -mediated resistance (e.g. Douchkov et al ., 2005; Zimmermann et al ., 2006) but still it is questionable whether the allelic variation in those candidate genes also determines natural variation in level of basal resistance between plant accessions.

5 Chapter 1

Non-host resistance The grass powdery mildew pathogen, Blumeria graminis, is classified into several formae speciales each colonizing an individual genus of the grass family. Blumeria graminis f.sp. hordei (Bgh ), the causal agent of powdery mildew disease on barley, for example, successfully colonizes cultivated barley Hordeum vulgare and its wild ancestor H. spontaneum but is probably not able to colonize other related species in the genus Hordeum nor other species in the Triticeae, such as wheat (Eshed & Wahl , 1970; Menzies & MacNeill, 1989). Conversely, cultivated barley cannot be colonized by powdery mildew isolates collected on other Hordeum species and other Triticeae. This resistance is known as non-host resistance, and is mainly associated with papilla formation at sites of attempted fungal penetration. A proportion of challenged epidermal cells show, however, a hypersensitive reaction. Although a similar mechanism of defense is involved in basal resistance of barley to the adapted pathogen, Bgh , at macroscopic and microscopic level a clear discrimination can be made between basal host resistance and non-host resistance, since in the latter, the resistance is complete. Since there is not any barley accession which is susceptible to the non-adapted mildew forms, like the wheat powdery mildew pathogen ( Bgt ), it is not known which genes determine the host status of barley to the non-adapted Bgt . mlo-mediated resistance Resistance mediated by the recessive loss-of-function mutant at the Mlo locus is race-non-specific, since it is effective against all known isolates of Bgh (Büschges et al ., 1997). Several resistance alleles of mlo have been derived from mutagenic treatment of susceptible wildtype Mlo barley cultivars, but the mlo resistance allele has also been identified from non-mutagenized landraces of barley from Ethiopia (Jørgensen, 1992). In mlo resistant plants, resistance at the penetration stage in association with papilla formation prevents the fungus to form haustoria in all kinds of epidermal cells, except the subsidiary cells of stomata. Papilla formation occurs also in Mlo susceptible plants when challenged by the penetration peg of the germinated powdery mildew conidium, but the penetration efficiency of Bgh is considerably higher in Mlo susceptible plants than in mlo resistant plants (Freialdenhoven et al ., 1996). On the other hand, papilla based responses occur earlier and the size of papillae are generally larger at the failed penetration sites in mlo resistant lines than at the sites of failed penetration in Mlo susceptible plants (Jørgensen , 1992). mlo-mediated resistance shows some level of pathogen specificity, since mlo alleles are not effective against other fungal pathogens including barley leaf rust ( Puccinia hordei ), stripe rust ( Puccinia striiformis ), stem rust ( Puccinia graminis ), scald

6 General introduction

(Rhynchosporium secalis ), and the take all fungal disease ( Gaeumannomyces graminis ) (Jørgensen, 1977; Collins et al., 2007). However mlo seems to play a role in non-host resistance of barley to the non-adapted mildew pathogen, Bgt (Peterhänsel et al ., 1997; Trujillo et al ., 2004; Humphry et al ., 2006). Mlo gene homologs have now been identified in a wide range of plants, including wheat, rice, tomato, pea, grapevine and the model plant Arabidopsis thaliana, some of which have been demonstrated to contribute to penetration resistance to powdery mildew (Elliott et al ., 2002; Consonni et al ., 2006; Fondevilla et al ., 2006; Bai et al ., 2008; Winterhagen et al ., 2008). mlo and non-host resistance share similar molecular components mlo and non-host resistance share many analogous features (Humphry et al ., 2006). 1- Both types of resistance are associated with strong penetration resistance based on papilla formation at the penetration sites. 2- Both mlo and non-host resistance are broad spectrum and durable. 3- They do not depend on Salicylic acid (SA), Jasmonic acid (JA) or Ethylene-mediated defense signaling pathways but both depend on actin cytoskeleton components. 4- Both types of resistance require PEN genes (Arabidopsis) or Ror genes (barley). 5- Both depend on the function of the SNAP34 protein. Cloning of the Mlo wild type allele, identification of Ror genes and characterization of Arabidopsis mutants with atenuated non-host interaction with Bgh contributed a lot to our understanding of the molecular basis of penetration resistance to powdery mildews (see Figure 2; Freialdenhoven et al ., 1996; Büschges et al ., 1997; Collins et al. , 2003; Consonni et al ., 2006). Two genes, Ror1 and Ror2 (required for mlo - specified resistance) are required for the function of mlo in barley. Mutation either in Ror1 or Ror2 decrease powdery mildew resistance in mlo genotypes (Freialdenhoven et al ., 1996). Peterhänsel et al ., (1997) showed that mlo and Ror genes are active when barley is challenged with Bgt , a non-adapted powdery mildew. Trujillo et al . (2004) indicated that mutants of Ror1 and Ror2 showed higher frequencies of fungal penetration in interaction with Bgt . Screening of mutagenized Arabidopsis populations for increased frequencies of penetration by the non-adapted powdery mildew fungus Bgh, resulted in identification of several penetration (pen ) mutants allowing higher frequencies of Bgh entry into epidermal cells of which three, designated pen1 , pen2 , and pen3 , have been characterized and the corresponding genes were identified (Collins et al. , 2003; Lipka et al. , 2005; Stein et al. , 2006). Cloning of PEN1 showed that it is a homologue of barley Ror2 and that this gene encodes SYP121, a syntaxin which is known to contribute to vesicle fusion events through the formation of SNARE

7 Chapter 1

complexes with a corresponding VAMP (vesicle associated membrane protein) and a SNAP25 homologue (Collins et al. , 2003). PEN2 encodes a glycoside hydrolase that is required for the enzymatic activation of small molecules and is involved in the production of an antifungal compound (Lipka et al. , 2005). PEN2 protein was found to be localized to peroxisomes, organelles that accumulate at the sites of attempted powdery mildew penetration (Koh et al ., 2005; Lipka et al ., 2005). PEN3 encodes an ATP-binding cassette (ABC) transporter protein (Stein et al ., 2006) possibly involved in exports of a potentially fungi-toxic compound that is processed by PEN2 in peroxisomes into the apoplast (Figure 2).

Figure 2. Schematic overview of molecular components involved in penetration resistance to powdery mildew. A fungal appressorium trying to breach the host cell wall by a penetration peg is stopped in a cell wall apposition (papilla). PEN1/Ror2 syntaxin and HvSNAP34 protein contribute to SNARE complex formation and secretion of antimicrobial compounds at sites of attempted fungal penetration. PEN3 is involved in export of potentially toxic compounds that were released by PEN2 enzyme activity in peroxisomes (reprinted by permission from Collins et al., 2007; http://www.publish.csiro.au/nid/40/paper/AR06065.htm ).

The possible functional link between mlo-mediated and non-host resistance was recently supported by identification of some candidate genes that emerged from transient overexpression or transient-gene silencing studies. These candidate genes were found to be required for mlo -mediated resistance against Bgh as well as for non-host resistance to Bgt (Douchkov et al ., 2005; Miklis et al ., 2007; Schweizer, 2007). Furthermore, overexpression of Mlo in epidermal cells of barley resulted in some degree of susceptibility to the non-adapted Bgt pathogen suggesting that Mlo may negatively regulate non-host resistance as well (Elliott et al ., 2002).

8 General introduction

Suppression and induction of penetration resistance Adapted pathogens have the ability to overcome preformed defense barriers and suppress induced defense responses, but non-adapted pathogens do not have such ability (Thordal-Christensen, 2003). Different fungal pathogens employ different suppression strategies to infect and colonize their hosts (Staples & Mayer, 2003). Biotrophic and haustorium forming fungal pathogens, like powdery mildews and rusts, first should suppress penetration resistance to be able to successfully penetrate through epidermal or mesophyll cell walls. Successful penetration and haustorium formation of virulent isolates of Blumeria graminis induces susceptibility to haustorium formation by subsequent challenge of either adapted or non-adapted powdery mildew pathogens. On the other hand, failed attempted penetration induces resistance to haustorium formation by subsequent challenge inoculation with a compatible isolate (e.g . Lyngkjær & Carver, 1999; Lyngkjær et al ., 2001; Kunoh, 2002; Olesen et al ., 2003). This induction of susceptibility and resistance at cell level is often called accessibility and inaccessibility. Studies on non-host interactions with cowpea rust fungus and plantain powdery mildew fungus suggests that the adhesion between the plant cell wall and the plasma membrane plays a role in cell wall-associated defense responses and powdery mildews and rust fungi employ different strategies to successfully induce accessibility of plant cells (Mellersh & Heath, 2001; Heath, 2002).

Scope and outline of the thesis In this thesis we investigated the genetic variation in barley for resistance to the adapted and non-adapted powdery mildew pathogens and we intend to identify the genes that are responsible for natural variation in host status and non-host resistance level. We aimed also to determine whether the basal resistance of barley to powdery mildew is being suppressed by prior attack by a different pathogenic haustorium forming fungus, Puccinia hordei that differs from the mildew fungus in its strategy to induce accessibility. In Chapter 2, a multi-population QTL analysis was conducted for powdery mildew resistance of barley at seedling and at adult plant stage. Twenty-three EST/cDNA- clone sequences were mapped that are likely to play a role in the barley-Blumeria interaction based on transcript profiling, gene-silencing or over-expression data, and mlo , Ror1 and Ror2. To compare the map position of QTLs over different mapping populations, we constructed an improved high-density integrated linkage map of barley, based on the marker data of seven mapping populations of barley. In Chapter 3, two experimental barley lines were developed with extremely high and low level of basal resistance to powdery mildew by convergent crossing between the

9 Chapter 1

most resistant and the most susceptible lines from four mapping populations of barley. In Chapter 4, a large collection of barley germplasm was screened and some rare barley accessions were identified with rudimentary susceptibility to the non-adapted wheat powdery mildew, Bgt . Those accessions were intercrossed in two cycles, and resulted in two lines, called SusBgt SC and SusBgt DC, with exceptional susceptibility to Bgt , that were tested with seven non-adapted forms of Blumeria graminis , to investigate the specificity of the non-host resistance of barley. In Chapter 5, the rust-induced powdery mildew resistance at the cellular level was characterized. We also identified differentially expressed genes that may explain the strong papilla-based penetration resistance in double inoculated barley plants. In Chapter 6, the results obtained in the previous Chapters, the importance of the experimental barley lines that were developed in this study for the identification of genes that determine basal resistance of barley to adapted and non-adapted Blumeria graminis forms is being discussed.

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Koh S, Andre A, Edwards H, Ehrhardt D, Somerville S. 2005 . Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections. The Plant Journal 44: 516–529. Kølster P, Munk L, Stølen O, Løhde J, 1986. Near-isogenic barley lines with genes for resistance to powdery mildew. Crop Science 26: 903–907. Kunoh H. 2002. Localized induction of accessibility and inaccessibility by powdery mildew. In: Bélanger RR, Bushnell WR, Dik AJ, Carver TLW, eds. The powdery mildews: A comprehensive treatise. St. Paul/Minnesota, USA: APS Press, 126–133. Lin MR, Edwards HH. 1974. Primary penetration process in powdery mildewed barley related to host cell age, cell type, and occurrence of basic staining material. New Phytologist 73: 131-137. Lipka V, Dittgen J, Bednarek P, Bhat R, Wiermer M, Stein M, Landtag J, Brandt W, Rosahl S, Scheel D, et al. 2005. Pre- and postinvasion defences both contribute to nonhost resistance in Arabidopsis. Science 310: 1180-1183. Lyngkjær MF, Carver TLW, Zeyen RJ. 2001. Virulent Blumeria graminis infection induces penetration susceptibility and suppresses race-specific hypersensitive resistance against avirulent attack in Mla1 -barley. Physiological and Molecular Plant Pathology 59: 243-256. Lyngkjær MF, Carver TLW. 1999. Induced accessibility and inaccessibility to Blumeria graminis f.sp. hordei in barley epidermal cells attacked by a compatible isolate. Physiological and Molecular Plant Pathology 55: 151–162. Lyngkjær MF, Newton AC, Atzema JL, Baker SJ. 2000. The barley mlo gene: an important powdery mildew resistance source. Agronomie 20: 745–756. Mellersh DG, Heath MC. 2001. Plasma membrane-cell wall adhesion is required for expression of plant defense responses during fungal penetration. Plant Cell 13: 413–424. Menzies JG, MacNeill BH. 1989. Infection of species of the Gramineae by Erysiphe graminis f.sp. hordei and Erysiphe graminis f.sp. tritici. Canadian Plant Disease Survey 69: 105-108. Miklis M, Consonni C, Bhat RA, Lipka V, Schulze-Lefert P, Panstruga R. 2007. Barley mlo modulates actin-dependent and actin-independent antifungal defense pathways at the cell periphery. Plant Physiology 144: 1132–1143. Olesen KL, Carver TLW, Lyngkjær MF . 2003. Fungal suppression of resistance against inappropriate Blumeria graminis formae speciales in barley, oat and wheat. Physiological and Molecular Plant Pathology 62: 37-50. Peterhänsel C, Freialdenhoven A, Kurth J, Kolsch R, Schulze-Lefert P. 1997. Interaction analyses of genes required for resistance responses to powdery mildew in barley reveal distinct pathways leading to leaf cell death. Plant Cell 9: 1397–1409. Schweizer P. 2007. Nonhost resistance of plants to powdery mildew-New opportunities to unravel the mystery. Physiological and Molecular Plant Pathology 70: 3–7. Staples RC, Mayer AM. 2003. Suppression of host resistance by fungal plant pathogens. Israel journal of plant sciences 51: 173-184. Stein M, Dittgen J, Sanchez-Rodriguez C, Hou BH, Molina A, Schulze-Lefert P, Lipka V, Somerville S. 2006. Arabidopsis PEN3/PDR8, an ATP cassette transporter, contributes to nonhost resistance to inappropriate pathogens that enter by direct penetration. Plant Cell 18: 1- 16. Stein N, Graner A. 2004. Map-based gene isolation in cereal genomes. In: Gupta PK, Varshney RK, eds. Cereal Genomics . Dordrecht, the Netherlands: Kluwer Academic Publishers, 331–360. Takamatsu S .2004. Phylogeny and evolution of the powdery mildew fungi (Erysiphales, Ascomycota) inferred from nuclear ribosomal DNA sequences. Mycoscience 45: 147-157.

12 General introduction

Thordal-Christensen H, Gregersen PL, Collinge DB. 2000. The barley/ Blumeria (syn. Erysiphe ) graminis interaction: a case study. In: Slusarenko AJ, Fraser RSS, van Loon LC, eds. Mechanisms of Resistance to Plant Diseases. Dordrecht, the Netherlands: Kluwer Academic Publishers, 77–100. Thordal-Christensen H. 2003. Fresh insights into processes of non-host resistance. Current Opinion in Plant Biology 6: 351-357. Trujillo M, Troeger M, Niks RE, Kogel KH, Hückelhoven R. 2004. Mechanistic and genetic overlap of barley host and non-host resistance to Blumeria graminis . Molecular Plant Pathology 5: 389- 396. Winterhagen P, Howard SF, Qiu W, Kovacs LG. 2008. Transcriptional up-regulation of grapevine MLO genes in response to powdery mildew infection. American Journal of Enology and Viticulture. 59: 159-168. Wyand RA. Brow, JKM. 2003. Genetic and forma specialis diversity in Blumeria graminis of cereals and its implications for host-pathogen co-evolution. Molecular Plant Pathology 4: 187-198. Zeyen RJ, Carver TLW, Lyngkjær MF. 2002. The formation and role of papillae. In: Bélanger RR, Bushnell WR, Dik AJ, Carver TLW, eds. The powdery mildews: A comprehensive treatise. St. Paul/Minnesota, USA: APS Press, 107-125. Zimmermann G, Baumlein H, Mock HP, Himmelbach A, Schweizer P. 2006. The multigene family encoding germin-like proteins of barley. Regulation and function in basal host resistance. Plant Physiology 142: 181-192.

13 Chapter 1

Which candidate genes are responsible for natural variation in basal resistance of barley to barley powdery mildew?

R. Aghnoum, T.C. Marcel, A. Johrde, N. Pecchioni, P. Schweizer and R.E. Niks

Submitted Chapter 2

Abstract Basal resistance of barley against powdery mildew is a quantitatively inherited trait that is based on non-hypersensitive mechanisms of defense. A functional genomic approach indicated many candidate genes to be involved in defense against fungal haustorium formation. It is not known for which of those candidate genes allelic variation may contribute to the natural variation for powdery mildew resistance, since many of them may be highly conserved within the barley species. Twenty-three EST/cDNA-clone sequences that are likely to play a role in the barley-Blumeria interaction based on transcript profiling, gene-silencing or over-expression data, and mlo , Ror1 and Ror2, were mapped and are considered candidate genes to contribute to basal resistance. We mapped QTLs for powdery mildew resistance in six mapping populations of barley at seedling and/or at adult plant stage and developed an improved high-density genetic integrated map containing 6,990 markers for comparing QTLs and candidate gene positions over mapping populations. We mapped 12 QTLs at seedling stage and 13 QTLs at adult plant stage of which four were in common between the two developmental stages. Six of the candidate genes showed coincidence in their map positions with the QTLs identified for basal resistance to powdery mildew. This co-localization justifies to give priority to those six candidate genes to verify them as being responsible for the phenotypic effects of the QTLs for basal resistance.

Introduction The barley-powdery mildew pathosystem is one of the best characterized and highly specialized systems of host-parasite interaction. More than 85 race-specific resistance genes to powdery mildew have been identified in barley (Jørgensen, 1994; Chelkowski et al ., 2003). They act through a hypersensitivity response (HR), which is only elicited by particular avirulent pathogen isolates according to the gene-for-gene concept (Collins et al ., 2002). Upon deployment of resistance genes in breeding programs, virtually all of these genes became ineffective (Czembor, 2000). Six major resistance genes of barley to powdery mildew ( Mla1 , Mla6, Mla7, Mla10, Mla12 , and Mla13 ) have been cloned and characterized (Chelkowski et al ., 2003; Stein & Graner, 2004). Another type of resistance is known as basal (partial) resistance and is conferred by many, mostly additively acting genes, each with a small effect. Basal resistance has been used to indicate a low-level penetration resistance expressed in 'susceptible' plants, which is not due to hypersensitivity (e.g. Collins et al., 2003). Since partial resistance is defined as a resistance characterized by a susceptible infection type

16 Which candidate genes are….

and a reduced disease severity (Parlevliet, 1975) we presume in this manuscript that partial and basal resistance in this pathosystem are synonymous terms. Basal resistance is considered as an important and potentially durable type of resistance. Nevertheless, it is the least investigated type of resistance. In barley-powdery mildew interaction, basal resistance is associated with formation of cell wall appositions, so- called papillae, at the fungal penetration sites and this reinforcement of cell walls prevents haustorium formation in the attacked epidermal cells. Failure of haustorium formation is also called “pre-haustorial” resistance and is in general not associated with a hypersensitive reaction of the challenged plant cells (Niks & Rubiales, 2002). Even the most susceptible barley genotypes prevent a substantial proportion of mildew infection units from forming their first haustorium, implying that all genotypes have at least some degree of basal resistance. Genotypic variation in level of basal resistance of barley to powdery mildew has been reported by several researchers (Asher & Thomas, 1984; Jones & Davies, 1985; Carver, 1986; Knudsen et al ., 1986; Mastebroek & Balkema-Boomstra, 1991). Histological studies on partially resistant genotypes showed that resistance to primary penetration by non-hypersensitive mechanisms affect a varying proportion (26-75%, dependent on barley accession) of attempted penetrations to fail (Asher & Thomas, 1987). Carver (1986) reported that in spring barley lines with various levels of basal resistance, a range from 48 to 90% of infection units failed at penetration stage. Development of molecular marker techniques has enabled researchers to dissect quantitatively inherited traits, like basal resistance, by QTL analysis and to study the effects of individual QTLs, including their interactions. In contrast to single and dominant major genes, the genetic and molecular basis of quantitative disease resistance is less well understood, because of the polygenic inheritance, the relatively small effect of individual genes and significant genotype × environment interactions that make such traits experimentally more difficult to assess. However, research is underway to isolate the genes underlying QTLs for basal resistance of barley to barley leaf rust Puccinia hordei following map-based cloning approaches (Marcel, 2007b). Such an approach would be feasible to identify the genes underlying the powdery mildew QTLs. The recessive allele of the Mlo -locus of barley mediates a race non-specific resistance to the barley powdery mildew. mlo resistance and basal resistance share the same mechanism of defense, i.e . both are “papilla-based” and “pre-haustorial” (Trujillo et al ., 2004; Schweizer, 2007). Mutational approaches revealed that two genes ( Ror1 and Ror2 ) are required for full expression of mlo resistance (Freialdenhoven et al ., 1996). These two genes have also been implicated in basal

17 Chapter 2

penetration resistance of barley expressed in susceptible ( Mlo ) genotypes (Collins et al ., 2003). So, mlo , Ror1 and Ror2 can be considered as candidate genes to be involved in basal resistance of barley to powdery mildew. Candidate gene analysis is based on the hypothesis that genes with known function could correspond to loci controlling traits of interest (Pflieger et al ., 2001). This approach has been widely applied for genetic dissections of complex and quantitative traits (Zhu & Zhao, 2007). For plant geneticists, a candidate gene is any gene putatively involved in trait variation, based on its biological function, its map position and/or its expression pattern (Pflieger et al ., 2001). Ideally, the list of candidate genes to explain a particular phenotype can be shortened by following a prioritarization approach, which consist in combining information on the function, position and expression of the genes involved (Zhu & Zhao, 2007). However, finally the candidate genes should be validated through physiological analyses or genetic transformation. To identify genes that influence barley-Blumeria interactions in host and non-host combinations, a high-throughput RNAi system for transient-induced gene silencing (TIGS) as well as a transient over-expression in single epidermal cells of barley has been developed (Schweizer et al ., 1999; Douchkov et al ., 2005; Ihlow et al ., 2008). Using this system, the function of 930 barley candidate genes including 693 up- regulated genes and 101 resistance-gene analogues expressed in barley epidermis have been tested for their effect on non-host resistance of barley to wheat powdery mildew and on the level of basal host resistance. The role of 43 genes in non-host resistance, basal resistance and/or ml o-mediated resistance of barley has been confirmed by using this method (Dong et al ., 2006; Zimmermann et al ., 2006; Johrde & Schweizer, 2008). Identification of candidate genes whose expression is correlated with haustorium formation as shown after overexpression or silencing does not necessarily mean that they are also responsible for natural variation in levels of basal resistance. Such candidate genes may simply be conserved within the plant population.The study of the inheritance of natural variation can be considered as an alternative approach to elucidate the functional role of candidate genes in a given phenotype (Mitchell-Olds & Schmitt, 2006). In the present study we mapped genes that determine natural variation in level of basal resistance in six barley mapping populations and compared their map positions with those of 23 markers derived from gene sequences that were found to be pathogen-induced or that produced a resistance phenotype upon RNAi or over- expression during the interaction of barley with powdery mildew and also mlo , Ror1 and Ror 2 genes. We combined the expression and function of candidate genes with

18 Which candidate genes are….

their map position in order to prioritize them. We also determined whether QTLs tended to map to major gene loci of resistance to powdery mildew.

Materials and Methods Marker data sets Segregating marker data from seven individual mapping populations (Table 1) were compiled to prepare a high-density genetic integrated map of barley. The populations consisted of four doubled haploids (DH) populations, i.e . Igri × Franka (IgFr, Graner et al., 1991), Steptoe × Morex (StMo, Kleinhofs et al., 1993), the Oregon Wolfe Barleys (OWBs, Costa et al., 2001) and Nure × Tremois (NuTr, Francia et al., 2004), and of three recombinant inbred lines (RIL) populations, i.e . L94 × Vada (LnVa, Qi et al., 1998), Vada × SusPtrit (VaSu, Jafary et al., 2006) and Cebada Capa × SusPtrit (CCSu, Jafary et al., 2008). We reused the data sets compiled by Marcel et al., (2007a) after excluding the AFLP markers mapped on the OWBs. We added the DArT (diversity arrays technology) and TDM (transcript-derived marker) marker data sets released by Wenzl et al., (2006) and Potokina et al., (2008) [StMo], respectively, the SSR and EST marker data sets released by Varshney et al., (2007) and Stein et al., (2007) [IgFr, StMo, OWBs], respectively, and new marker data posted on the Oregon State University (OSU) Barley Project web site (http://www.barleyworld.org/ ) [OWBs]. The marker data set of NuTr consisted of 542 markers, mostly DArT markers (72%), and was obtained from Dr. Nicola Pecchioni (Università di Modena e Reggio Emilia, Italy). Segregating data from most of the BIN markers defined by Kleinhofs & Graner (2001) [StMo] were obtained from Dr. Andris Kleinhofs (Washington State University). Three new marker data sets were also included in the present integrated map and consist of 168 “PERO” markers mapped in LnVa and VaSu by peroxidase-profiling (to be published elsewhere by our laboratory), 26 new EST-SSR markers mapped in NuTr or CCSu by A/T labelling (Marcel et al., 2007a), and the 23 candidate genes described in the present study (Table 4).

Construction of the barley integrated map The integrated map was constructed essentially as described in Marcel et al., (2007a) using the software JoinMap 4 TM and MapChart (Voorrips, 2002). All the bridge markers ( i.e. markers common to two or more populations), the 210 BIN markers defined by Marcel et al., (2007a) and the additional BIN markers provided by Dr. A. Kleinhofs were used to prepare the framework map. The order of the 210 BIN markers was initially used as fixed order to run the regression mapping algorithm.

19 Chapter 2

After each mapping, the obtained marker order was compared with the marker order on the integrated map of Marcel et al., (2007a). Most discrepancies between the two marker orders were investigated by graphical genotyping in order to reject or to validate the newly calculated map. In cases where the newly calculated map was rejected, the fixed order was modified and the mapping repeated until the discrepancy was corrected. Consequently, in cases where the new map was validated, it could only represent an improvement from the previous integrated map of Marcel et al., (2007a). Then, the marker order on the constructed framework map was used as fixed order to run the maximum likelihood mapping algorithm on the 49 linkage groups ( i.e . seven barley chromosomes for seven mapping populations); default values were increased to five map optimization rounds with 3,000 chains. Three maps were generated for each linkage group and aligned to compare the order and distances between markers. From each alignment/linkage group, the map showing the most consistent marker order and the shortest total length was integrated into the framework map following the “neighbors” approach. The obtained integrated map was divided into 217 BINs of about 5 cM each. The BIN-defining markers of Kleinhofs & Graner (2001) were maintained in their role while the BIN- defining markers of Marcel et al ., (2007a) were sometimes modified to obtain a more homogeneous distribution of the BINs over the map.

Plant and pathogen materials Six out of seven (IgFr excluded) mapping populations were evaluated for their level of basal resistance to barley powdery mildew at seedling stage under controlled conditions and five of them (IgFr and OWBs excluded) at adult plant stage under field conditions. An isolate of Blumeria graminis f.sp. hordei collected in Wageningen 2004 (Wag.04) was used for evaluation of resistance levels at seedling stage. This isolate was maintained in a climate cabinet and propagated on young seedlings of the susceptible barley cultivar ‘Manchuria’. Table 1. Characteristics of the seven barley mapping populations used to construct the integrated map of which six were used for QTL analysis.

Name of population Type Lines Number of markers

L94 × Vada (LnVa) RIL 103 1083 Vada × SusPtrit (VaSu) RIL 152 533 C.Capa × SusPtrit (CCSu) RIL 113 519 OWBs DH 94 882 Steptoe × Morex (StMo) DH 150 3561 Nure × Tremois (NuTr) DH 118 542 Igri × Franka (IgFr) DH 71 737

20 Which candidate genes are….

Table 2 . Comparison of several (integrated) linkage maps published for barley Map length Number Average locus number per cM Contributing Populations 1 (cM) of loci

1H 2H 3H 4H 5H 6H 7H Rostoks et al ., 1,211 1,237 0.9 1.1 1.0 1.1 0.9 1.0 1.2 LiHs, OWBs, StMo 2005 Wenzl et al ., 2006 1,161 2,935 2.8 3.1 2.5 1.9 2.0 2.5 3.0 BaCP, ClSa, DaZh, FoCI, FrSt, IgAt, PaTa, StMo, TXFr, YeFr Marcel et al ., 2007 1,081 3,258 2.6 3.7 3.3 2.4 2.3 2.9 3.8 LnVa, VaSu, StMo, OWBs, IgFr, CCSu Stein et al ., 2007 1,118 1,055 1.0 1.1 0.9 0.8 0.9 0.8 1.2 StMo, OWBs, IgFr Potokina et al ., 1,010 1,596 1.3 1.8 1.9 1.3 1.4 1.5 1.7 StMo 2008 This report 1,093 6,990 6.4 7.9 6.9 4.5 5.7 5.9 7.3 LnVa, VaSu, StMo, OWBs, IgFr, CCSu, NuTr

1 LiHs (Lina x Hordeum spontaneum HS92); OWBs (Oregon Wolfe Barleys); StMo (Steptoe x Morex); BaCP (Barque-73 x CPI71284-48); ClSa (Clipper x Sahara); DaZh (Dayton x Zhepi2); FoCI (Foster x CI4196); FrSt (Fredickson x Stander); IgAt (Igri x Atlas68); PaTa (Patty x Tallon); TXFr (TX9425 X Franklin); YeFr (Yerong x Franklin); LnVa (L94 x Vada); VaSu (Vada x SusPtrit); IgFr (Igri x Franka); CCSu (Cabada Capa x SusPtrit); NuTr (Nure x Tremois)

21 Chapter 2

Assessment of resistance level at seedling stage Evaluation of powdery mildew resistance at seedling stage was performed on detached leaves (Schwarzbach, 2004). Barley plants were grown in spore-free greenhouse compartments. Twelve days after sowing, when the first leaf was fully expanded, a segment of about 4 cm long was cut from the middle part of the first leaves, and both cut ends were immersed in 0.6% water agar medium containing 125 ppm benzimidazole in a square (12 × 12 cm) polystyrene petri dish, the adaxial side of the leaf facing upwards. Each petri dish accommodated about 16 leaf segments. One leaf segment was taken per RIL/DH line per experiment. Both parents of each mapping population and the susceptible cultivar ‘Manchuria’ were added to each petri dish as reference. To use freshly produced conidia for inoculation, young seedlings of the susceptible cultivar ‘Manchuria’ were densely inoculated with powdery mildew 7 days prior to inoculation of the experiment. A settling tower was used for inoculations (Schwarzbach, 2004). Per inoculation, four petri dishes containing the leaf segments were put inside the tower and conidia from heavily infected leaves were blown into the tower using compressed air and were allowed to settle for 10 minutes. The density of inoculum was monitored by haemocytometer and was adjusted to 10-15 conidia per mm 2. Following inoculation, the closed petri dishes were incubated in a growth chamber at 20 oC with fluorescent light irradiance (102.8 micromol/m 2/s) supplied for 12 h per day. Four days after incubation, the accessions were scored by counting the number of young powdery mildew colonies per 2 cm 2 of leaf segment, using a magnifying glass (10x). The experiment was repeated three times and the average infection frequency used for QTL analysis. Because L94 is a mlo -barley and fully resistant to powdery mildew, in our inoculation experiment we only included non- mlo carrying RILs (51 accessions) of the LnVa population (Shtaya et al ., 2006).

Histology of infection by powdery mildew To determine the mechanism of resistance at the cellular level, one DH line or RIL from each mapping population showing high levels of basal resistance at seedling stage together with the parents were selected for microscopic observation. The susceptible barley cultivar ‘Manchuria’ was included as reference. From each accession two seedlings were grown in boxes (37 × 39 cm). Twelve days after sowing, first formed leaves were fixed in a horizontal position, with the adaxial side up. The inoculation was performed using the settling tower with freshly produced spores collected from young susceptible barley plants. The inoculum density was adjusted to 10-15 conidia per mm 2. In each inoculation, one seedling from each accession was sampled for histological analysis and the other one was kept for

22 Which candidate genes are….

checking the efficiency of inoculation. The experiment was performed in two replications. Forty eight hours after inoculation (hai) a leaf segment from the inoculated leaves was cut and transferred to a solution of 1 mg.ml−1 3,3'- Diaminobenzidine (DAB) for 8 h and, subsequently, to a solution of acetic acid- ethanol (3/1 v/v) to be cleared overnight. The leaf segments were stained with 0.005% Trypan Blue as described by Anker & Niks (2001). To make the preparations, the leaf segments were embedded adaxial side up in glycerol. Leaf segments were examined at 400x magnification using an Axiophot microscope (Zeiss, Germany). In all microscopic observations only interstomatal (type B epidermal cells, following the terminology proposed by Koga et al ., 1990) were examined. Those germlings that were stopped by papillae at penetration stage were considered as failed primary penetration (FPP) and germlings that formed a haustorium and elongated secondary hyphae (ESH) were considered as successful primary penetration (SPP) (Asher & Thomas, 1983). Those epidermal cells that were challenged by more than one conidium were not considered.

Phenotypic assessment at adult plant stage Evaluation of basal resistance at adult plant stage was carried out at field conditions under naturally occurring powdery mildew infections. The parents and lines of different mapping populations were sown in three replications according to a randomized complete block design in spring of 2006 and 2007. Each genotype was sown in three-row plots of one meter long, row distance 25 cm. To minimize the effect of interplot interference (Parlevliet & Van Ommeren, 1984; Lipps & Madden , 1992), three rows of oat were sown between two neighbouring genotypes. The plots were treated following standard agricultural practices. Mildew severity was recorded two or three times with 8-10 days interval according to the 0-9 scale (0= no infection and 9= fully covered by mildew) (Saari & Prescott, 1975). The area under the disease- progress curve (AUDPC) was calculated (Jeger & Viljanen-Rollinson, 2001) and used as quantitative trait for QTL analysis. For the CCSu population, in the 2006 field test, disease severity was recorded only once and directly used for QTL analysis. For NuTr, the data were collected from 2 × 3 meter yield trial plots of 88 spring lines during the spring of 2005 and 2006, without alternating oat plots. The possible effects of interplot interference were minimized by sampling only the middle rows of the plots.

QTL analysis The quantitative resistance data were combined with the already available biparental marker map data and analyzed with the software package MapQTL version 5 (Van

23 Chapter 2

Ooijen, 2004). First an interval mapping (IM) analysis was performed. Markers with the highest log-likelihood ratios (LOD value) were selected as the initial set of possible co-factors for multiple-QTL mapping (MQM) analysis (Jansen, 1993; Jansen & Stam, 1994). The LOD threshold for detecting QTLs was calculated by a permutation test with 10000 iterations at the significance level α = 0.05. A threshold LOD value above or equal to the LOD value suggested by the permutation test was used to declare the presence of a QTL. Detected QTLs were transferred from the individual maps to the barley integrated map. QTLs were named as ‘ Rbgq ’ (quantitative genes for Resistance to Blumeria graminis ) followed by the QTL number as suggested by Shtaya et al ., (2006). A common name is given to QTLs for which the LOD-1 interval was overlapping.

Mapping candidate genes and their co-localization with QTLs Thirty four EST-derived sequences were provided by Dr. Patrick Schweizer (The Leibniz Institute of Plant Genetics and Crop Plant Research, IPK, Germany) and 5 sequences by Dr. Ralph Hückelhoven (Technical University of Munich, Germany). These ESTs represent candidate genes implicated in basal resistance, since they promoted or decreased haustorium formation by Blumeria graminis upon RNAi silencing or over-expression. We tried to convert those sequences to polymorphic PCR markers to map them on one of the barley mapping populations used to construct the integrated map. Primer pairs were designed using DNASTAR software package based on the full-coding-sequence available or using databases of genomic or cDNA sequences (CR-EST: The IPK Crop EST Database; The National Centre for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/ and The TIGR Gene Index at DFCI). EST sequences were converted to Cleaved Amplified Polymorphic Sequence (CAPS), Derived Cleaved Amplified Polymorphic Sequences (dCAPS) or Sequence Characterized Amplified Region (SCAR) markers to be mapped in one of the four mapping populations: CCSu, OWBs, StMo and LnVa. The candidate gene based markers were placed on the integrated map of barley to be compared with the position of QTLs for resistance to powdery mildew. We considered mlo and the two genes that are required for the mlo -mediated resistance, Ror1 and Ror2 , as candidate genes to compare their map positions with QTLs. mlo gene was already mapped directly in the LnVa population and was present in our integrated map. We located the Ror genes on the map based on the position of reported co-segregating markers (Collins et al ., 2001; Collins et al ., 2003). To determine the global significance of co-localizations between QTLs and candidate genes, a chi-square test was performed. The null hypothesis of independent

24 Which candidate genes are….

distribution of 5 cM BINs containing a QTL peak marker and BINs containing a candidate gene was tested.

Comparing the map position of QTLs with known R genes The Mla6 and MlLa genes were already located on the integrated map based on the marker data of IgFr and LnVa populations. Other R genes ( Mlra , Mlk , Mlnn , Mlga , Mlg , Mlj , mlt , Mlf ) were located on the map based on the reported position of co- segregating markers (Schonfeld et al ., 1996; Kurth et al ., 2001; Jahoor et al., 2004).

Results The new barley integrated map The new barley integrated map contains 6,990 morphological/molecular markers covering a total genetic distance of 1,093 cM, which is similar to previously reported barley integrated genetic maps (Table 2). The framework map contained 94 (for chromosome 1H), 149 (2H), 131 (3H), 85 (4H), 126 (5H), 116 (6H) and 122 (7H) (= 823 in total) bridge markers and covers a genetic distance of 1,051 cM. The most represented types of molecular markers on this integrated map are RFLP (20%), AFLP (20%), SSR (9%), DArT (19%) and TDM (23%) (= 91% of all markers). RFLP and AFLP marker techniques are not used so commonly anymore but the integration of such markers with more modern types of markers allow to integrate past and present data collected on many different mapping populations as well as to select markers or candidate genes linked to a specific trait in any of these populations. More than 43% of the markers on the present barley integrated map target ESTs or gene sequences (gene-targeted markers, GTM) and can be used for candidate gene identification (Rostoks et al., 2005; Marcel et al., 2007a) or to perform synteny analyses (Rostoks et al., 2005; Stein et al., 2007). The StMo map contributed more than half of the total number of markers (3,561 markers) on the integrated map, most of these markers coming from the recently released array-based marker data sets (Wenzl et al ., 2006; Potokina et al., 2008). The very low percentage of errors and high quality of these marker data greatly increase the reliability of our integrated map. The present integrated linkage map was aligned with the one of Marcel et al., (2007a) and with the TDM-based map of Potokina et al ., (2008) in order to compare their locus order. The marker order between the present integrated map and the TDM- based map was highly consistent, with only a few and very localized inversions pinpointing small inaccuracies inherent to map integration projects (Jackson et al., 2008). The marker order was also consistent with the integrated map of Marcel et al., (2007a) with the exception of reordering between several AFLP markers mapped at

25 Chapter 2

the telomeres of chromosomes 5HL and 6HL in the CCSu population. The inspection of graphical genotypes mostly agreed with the marker order on the present integrated map. The constructed barley integrated map has been posted on the GrainGenes website (Barley, Integrated 2008, Marcel" at http://wheat.pw.usda.gov/ ).

QTLs mapped at seedling stage The infection frequency of powdery mildew in detached seedling leaf tests exhibited continuous variation in all six mapping populations indicating quantitative inheritance. In all tested mapping populations, transgressive segregation towards resistance and towards susceptibility was observed (Figure 1), indicating that the resistance and susceptibility alleles were contributed by both parents. The number, genomic position on the barley integrated map, and effect of QTLs associated with basal resistance to powdery mildew at seedling stage are summarized in Table 3 and Figure 2. In total, 12 QTLs were mapped that affect the infection frequency at seedling stage in six mapping populations. The mapped QTLs were found on all chromosomes. One QTL on chromosome 5H ( Rbgq15 ) was in common between VaSu and LnVa populations. The phenotypic variation explained by the QTLs ranged from 6.2-24.8%. The LOD scores of QTLs were generally low (≤6.1). Our results indicate a high level of diversity in QTLs to be involved in powdery mildew resistance at seedling stage in barley.

QTLs mapped at adult plant stage The RIL and DH populations showed a continuous distribution for AUDPC or disease severity data collected at field experiments. Transgressive segregation was common in four out of five (StMo, no transgression) populations tested (Figure 3). In total, 13 QTLs for adult plant resistance, including QTLs mapped in different populations at the same location, were detected and these were distributed over all barley chromosomes (Table 3 and Figure 2). Three out of the 13 QTLs ( Rbgq7, Rbgq8 and Rbgq9 ) were mapped in more than one population. Four QTLs were detected in both developmental stages ( Rbgq2a, Rbgq7, Rbgq9, and Rbgq13 ) of which two were detected in the same population at seedling and adult plant stages. The phenotypic variation explained by the QTLs ranged from 7.3% to 52.1%, indicating great differences in effect size of QTLs conferring resistance at adult plant stage. Four QTLs ( Rbgq12 , Rbgq4 , Rbgq7 and Rbgq8 ) were detected and confirmed in both years. The most consistent QTLs were also the ones with the highest LOD scores (Table 3).The inconsistency of the other QTLs may be due to a high level of environment dependency or due to different race composition of the pathogen

26 Which candidate genes are….

population between years. Our results indicate that most QTLs for powdery mildew resistance in barley are highly plant development stage dependent and that a high level of genetic diversity exists in barley for basal resistance to powdery mildew.

OWBs-seedling stage NuTr-seedling stage

30 40 Tremois Dom Rec

30 Nure 20

10 10 Number of individuals Number of Number of individuals Number of

0 0 <=20 21-40 41-60 61-80 81-100 >100 <=20 21-40 41-60 61-80 81-100 >100 Infection frequency Infection frequency

LnVa-seedling stage StMo-Seedling Stage 30 50 Vada Morex 40 20 30 Steptoe

10 20

Number of individuals Number of 10 Numberof individuals 0 0 <=20 21-40 41-60 61-80 81-100 >100 <=20 21-40 41-60 61-80 81-100 >100 Infection frequency Infection frequency

CCSu-seedling stage VaSu-seedling stage

70 60 SusPtrit SusPtrit C.capa 60 50 Vada 50 40 40 30 30 20 20

Number Number of individuals 10 Number of individuals Number of 10 0 0 <=20 21-40 41-60 61-80 81-100 >100 <=20 21-40 41-60 61-80 81-100 >100 Infection frequency Infection frequency

Figure 1. Histograms of the frequency distribution of powdery mildew resistance phenotypes in six mapping populations of barley tested at seedling stage. Infection frequency represents the number of colonies in 2 cm 2 of infected leaf segments 4 days after inoculation.

27 Chapter 2

Table 3. Characteristics of QTLs mapped in six barley populations against powdery mildew ( Blumeria graminis f.sp. hordei) QTL Growth Population Chrom. Peak marker Position LOD % exp. a Add. b Donor c Name stage (PM) of PM

Rbgq4 Field2005 NuTr 1H bPb-4657 23.8 7.2 28.4 10.05 Tremois

Rbgq8 Field2005 NuTr 2H bPb-2230 54.3 6.0 22.1 9.10 Tremois

Rbgq12 Field2005 NuTr 4H E39M61-181 71.6 6.3 31.2 10.66 Tremois

Rbgq4 Field2006 NuTr 1H bPb-4657 23.8 9.6 37.3 6.35 Tremois

Rbgq7 Field2006 LnVa 2H E41M32-83 32.3 7.3 52.1 17.08 Vada

Rbgq21 Field2006 VaSu 7H P15M47-184 40.9 3.3 8.6 0.58 SusPtrit

Rbgq8 Field2006 LnVa 2H E33M54-187 57.0 3.3 27.5 11.86 Vada

Rbgq18 Field2006 CCSu 6H E33M55-333 58.5 2.8 10.4 0.37 Cebada Capa

Rbgq8 Field2006 NuTr 2H bPb-6881 59.2 6.0 19.1 4.66 Tremois

Rbgq8 Field2006 VaSu 2H E33M54-182 61.1 7.7 20.0 0.85 Vada

Rbgq12 Field2006 NuTr 4H E39M61-181 71.6 4.9 37.1 6.34 Tremois

Rbgq9 Field2006 LnVa 2H E38M54-294 147.6 4.7 34.7 13.03 Vada

Rbgq9 Field2006 VaSu 2H P17M54-152 148.4 3.6 9.9 0.60 Vada

Rbgq7 Field2007 LnVa 2H E41M32-83 32.3 3.1 16.1 3.40 Vada

Rbgq7 Field2007 StMo 2H MWG858 37.8 8.7 17.2 2.33 Steptoe

Rbgq5 Field2007 StMo 1H ABG053 43.1 5.7 10.9 1.89 Morex

Rbgq8 Field2007 CCSu 2H E38M54-169 54.3 3.4 14.3 4.00 Cebada Capa

Rbgq8 Field2007 VaSu 2H E38M54-169 54.3 13.3 31.1 4.08 Vada

28 Which candidate genes are….

Table 3 continued QTL Growth Peak marker Position of Population Chrom. LOD % exp. a Add. b Donor c Name stage (PM) PM

Rbgq2a Field2007 LnVa 3H E33M61-131 63.8 4.3 24.9 4.27 L94 Rbgq11 Field2007 StMo 3H CDO113B 106.7 3.6 7.3 1.49 Morex Rbgq13 Field2007 VaSu 4H E41M40-79 138.3 3.9 7.9 2.07 SusPtrit Rbgq16 Field2007 StMo 5H CDO484 161.1 4.2 7.6 1.55 Morex Rbgq17 Field2007 VaSu 5H E33M61-144 178.0 4.8 9.5 2.22 SusPtrit Rbgq20 Seedling OWBs 7H ABG704 7.4 3.5 18.3 11.12 Rec Rbgq10 Seedling StMo 3H MWG571C 16.4 4.7 15.5 9.90 Morex Rbgq7 Seedling CCSu 2H E37M32-288 25.5 3.8 14.4 6.24 SusPtrit Rbgq14 Seedling OWBs 5H Bmac0047e 44.9 4.9 19.9 11.40 Dom Rbgq2a Seedling NuTr 3H bPb-0312 47.7 6.1 24.8 9.36 Tremois Rbgq22 Seedling CCSu 7H E38M61-128 48.7 4.2 20.0 8.68 SusPtrit Rbgq6 Seedling VaSu 1H E38M54-441 61.5 3.4 8.0 6.07 SusPtrit Rbgq2b Seedling LnVa 3H E35M48-410a 69.5 2.8 19.8 9.98 L94 Rbgq19 Seedling NuTr 6H scssr05599 96.9 5.6 22.4 8.76 Nure Rbgq15 Seedling VaSu 5H E42M48-282 115.4 2.7 6.2 5.18 SusPtrit Rbgq15 Seedling LnVa 5H E33M58-306 117.6 3.1 21.5 10.71 Vada

Rbgq13 Seedling VaSu 4H E35M58-56 144.0 4.2 9.8 6.53 SusPtrit

Rbgq9 Seedling VaSu 2H E42M48-405 146.4 5.0 11.5 6.97 Vada a The proportion of the phenotypic variance explained by the QT b c Effects of the resistant allele in reduction of the number of colonies per 2 cm 2; Donor of the resistance allele

29 Chapter 2

1H 2H 3H

0 *BCD1434 *MWG844A *BCD907

*ABA305 *GBR0613 5 Rbgq10(Morex)_4.7

*E42M51-113 *ABG703B 10 GBM1447 *E37M33-160 WBE212 Rbgq7(SusPtrit)_3.8 *MWG938 Rbgq4(Tremois)_9.6 *ABG070 15 Mlra Rbgq4(Tremois)_7.2 *MWG878 *JS195F

*cMWG645 Rbgq7(Vada)_3.1 20 *MWG837 *E35M48-250 *GBS0535 25 Mla6 Rbgq7(Vada)_7.3 Mlk GBM5023 Rbgq7(Steptoe)_8.7 *E42M55-233 30 *GBM1007 Rbgq5(Morex)_5.7 GBM5018 *ABA004 *ABG318 *ABG321 35 *GBR1154 *E33M61-135 GBM1430 GBM5140 Rbgq2a(Tremois)_6.1 *BCD098 *ABG358 *MWG798B 40

*GBR1550 Rbgq8(Tremois)_6.0 *ABG053 *MWG584

WBE226 Rbgq8(Vada)_13.3 45 Mlnn GBM1284 *Pox Rbgq8(Tremois)_6.0 Rbgq8(C.Capa)_3.4 *Ica1 GBM5095 *BCD153 *E38M54-169 50 Rbgq8(Vada)_7.7 *E35M61-92 GBM1446 Rbgq2a(L94)_4.3 *Bgq60 *E37M32-471 Rbgq8(Vada)_3.3

55 Rbgq6(SusPtrit)_3.4 *JS074 GBM1158 GBM1412 GBM1172 60 Ror1 GBM1203 *ABG396 GBM1487 GBM1366 *Pcr2 Rbgq2b(L94)_2.8 65 GBM1129 *GBM1372 GBM1336 *GBM5230 70 GBM1216 *MWG571B GBM1480 WBE219 *ABC468 GBM1163 *bPb-1193 75 *Glb1 WBE231 *ABC176 GBM1153 *GBMS160 GBM5020 80 *GBM1108 GBM1513 GBM1343 *ABC451 WBE223 *ABG377 *MWG865 *DAK123B WBE232 85 GBM1468 Mlga Contig7079_at GBM1408 GBM1481 Contig7080_at 90 *Bmag0125 *GBR0643 GBM1090 *PSR330 WBE229 GBM5047 95 *Bmac0144a *MWG503 *MWG555B GBM5162 GBM1328 *ABG453 Rbgq11(Morex)_3.6 100 *cMWG649B GBM1440 *ABR320 *MWG882 *CDO345 *cMWG706A *GBM1208 *bPt-7238 105 *ksuD22 *CDO113B *ABC257 GBM1469 110 *E33M54-307 *GBS0510 *BCD193 *His4B 115 *GBS0379 GBM1437 GBM1226 *GBR1418 *MWG847 120 *ABC252 GBM1200 *ABG702 *ABG004 125 *ABC261 *GBM1047 WBE221 *WG110 130 GBM1308 *ABC165A *GBR0828 *ABC161 135 Rbgq9(Vada)_4.7 GBM1118 GBM1358 WBE220 Rbgq9(Vada)_5.0

GBM1407 *E35M61-358 Rbgq9(Vada)_3.6 *MWG902 140 *GBR1390 WBE211 GBM1314 *ABC174 *MWG844C 145 WBE225 GBM1288 150 MlLa WBE218 *MWG2200 *Adh10

155 GBM5080 *ABC166 160 *GBR1425 165 *ABC172 170 GBM1420

175 180

Figure 2. Localization of candidate gene markers and QTLs for resistance to powdery mildew on an integrated map of barley. The candidate genes, including mlo , Ror1 and Ror2 genes, are in bold red. The locations of QTLs are indicated by vertical bars at the right side of each linkage group. Solid bars represent QTLs identified at seedling stage under controlled conditions, and shaded bars represent QTLs identified at adult plant stage under field conditions . The length of a bar approximately represents the LOD-1 support interval and the length of the exceeding line the LOD-2 support interval of the QTL based on rMQM results. The ruler indicates the distance in cM. (Continued on next page)

30 Which candidate genes are….

4H 5H 6H 7H

0 *bPt-7602 *bPb-2330 *ABG062 *E37M50-401

*DAK133 Rbgq20(Rec)_3.5 5 *ABG466 *bPt-7852 *ABG704 10 mlt *ABR313 *ABG378 *MWG634 *MWG920-1A *bPt-3623 15 *E42M54-160 *bPb-0091 *ABG320 20 *E35M54-548 *E33M61-401 *scssr07106 WBE224 25 *JS103.3 *E45M55-400 *cMWG652A *P14M61-73

GBM5028 Rbgq21(SusPtrit)_3.3 *ABG705

Rbgq14(Dom)_4.9 *ABC151A 30 WBE227 *E33M55-63 *HVM40 WBE217 *CDO669B GBM1270 *GBM1326 35 GBM1252 Contig5975_at *DD1.1C GBM5060 *Ole1 Contig5974_s_at *ABG380 40 *LoxA WBE100 *ABG395 *ABG387B

*bPt-6944 Rbgq22(SusPtrit)_4.2 *E35M61-269 Rbgq12(Tremois)_4.9

GBM1478 Rbgq18(C.Capa)_2.8 45 *BCD402B *E32M62-321 GBM5086 GBM1222 GBR0504 *ksuA1A 50 *HVKNOX3 *GBM1053 Rbgq12(Tremois)_6.3 GBM1325 GBM1508 *iLdh1 *GBM1464 55 *BCD808B *Ltp1 GBM1267 *GBMS196 WBE215 GBM5226 Mlg 60 GBM1293 GBM5012 *Brz *ABC303 *WG530 *GBS0468 *ABG484 65 *E35M55-181 *ABG474 *ABC255 GBM1364 *GBS0042 *PBI25 *ABC302 *GBMS180 *HVCMA 70 GBM1506 *bBE54A *E33M54-261 *E38M55-128 *ABC170B *ABG701 75 Rbgq19(Nure)_5.6 GBM1237 *BCD926 *GBS0767 80 *Bmac0310 *ABC455 GBM5210 WBE101 *Nar7 Contig2163_at 85 *BCD453B *MWG522 Contig2170_at *GBM1350 Mlf

Rbgq15(SusPtrit)_2.7 90 *ABG473 *GBR0621 *Amy2 Mlj GBM1303 *ABG319A *GBM1227 95 *MWG934 WBE210 *E39M61-367 GBR0180 GBM1472 100 *E42M51-335 GBM1297 GBM1249 *MWG2230 *Tef1 GBM1396 GBM1295 105 *KFP221 GBM1428 GBM1141 *scssr00103 *GBMS141 Rbgq15(Vada)_3.1 WBE216 *GBS0531 *RZ242 110 *bPb-1325 WBE228 *Bmac0040a GBM5225 mlo GBM5008 *bPb-9908 115 *ABG397 *MWG514B *ABC310B *P16M51-215 GBM5222 *bPb-9104 GBM1154 *ABC305 120 *GBM1448 GBM5212 GBM1384 GBM5064 *GBR1547 GBM5214 *GBM1274 GBM1362 125 WBE214 Rbgq13(SusPtrit)_3.9 Rbgq13(SusPtrit)_4.2 GBM1275 WBE230 *ABG319C *GBS0138 *WG908 GBM1276 *ABG461A 130 *MWG2037B GBM1166 *MWG2196 GBM1324 Ror2 *cMWG684A *GBR1326 135 GBM1147 *E42M55-75 WBE104 *GBM1456 140 *E42M58-312 *ABG496 GBM1453 GBM1490 *GBM1018

Rbgq16(Morex)_4.2 145 *GBR1567 *GBS0800 150 *bPb-1858 *ABG391 155 *ABG390

160 Rbgq17(SusPtrit)_4.8 *DsT-33 165 *ABG463 170 *ABC309 175 GBM1474 *MWG851B 180

31 Chapter 2

LnVa-Field 2006 NuTr-Field 2006 30 80 70 60 20 Tremois 50 40 Vada 10 30 20 Number Number of individuals

Number of individuals 10 0 0 <=20 21-40 41-60 61-80 81-100 >100 <=50 51-70 71-90 91-110 >110 AUDPC AUDPC

StMo-Field 2007

CCSu-Field2007 90 70 80 60 70

50 SusPtrit 60 C.Capa 50 40 Steptoe 40 30 30 20 Morex 20

Number of individuals Number 10 Number10 of individuals 0 0 <=20 21-40 41-60 61-80 81-100 >100 <=20 21-30 31-40 41-50 >50 AUDPC AUDPC

VaSu-Field 2007

90 80 Vada 70 SusPtrit 60 50 40 30 20 Number of individuals of Number 10 0 <=20 21-30 31-40 41-50 >50 AUDPC

Fig ure 3 . Histograms of the frequency distribution of powdery mildew resistance phenotypes in 5 mapping populations of barley tested at adult plant stage in field conditions.

32 Which candidate genes are….

100% 90% 80% 70% 60% SPP-HR 50% SPP+HR 40% FPP 30% Interaction Interaction sites 20% 10% 0% Rec L94 Dom Nure Vada Morex Steptoe CCapa SusPtrit Tremois Manchuria StMo 39( R) StMo OWB 34( R) OWB CCSu 92( R) CCSu LnVa 26 R) ( LnVa VaSu 111( VaSu R) NuTr 122( R) NuTr

Figure 4. Defense reaction of the parents and most resistant line of different mapping populations to Bgh at 48 hai. Data of each group represent the mean of two leaf segments; in each leaf 100 infection sites have been evaluated. Gray-dotted column (FPP) represent failed primary penetration where the fungus was stopped at penetration stage; Black column (SPP+HR), successful primary penetration resulting in a haustorium but showed HR; White column (SPP-HR), successful primary penetration with haustorium and without HR. The lines with the (R) represent the most resistant line from each mapping population.

Histology of resistance at seedling stage The reaction to Bgh at 48 hai are presented for the parental lines and for the most resistant lines of each of the six studied mapping populations in Figure 4. At macroscopic level, all parental lines, except L94 that carries the mlo gene, showed a compatible infection type (infection type 4). This implies that they do not carry an effective race-specific resistance gene ( R gene) to the isolate Wag.04 that was used. The proportion of failed infection units that were stopped at penetration stage (pre- haustorial resistance) in parental lines ranged from 20% (Rec) to 63% (SusPtrit) of attempted penetration sites. Always a higher proportion of infection units were stopped at penetration stage in the most resistant lines rather than in the parents of the mapping populations. Apart from the mlo -caused pre-haustorial resistance in L94, the highest proportion of papilla-based resistance (83 %) was in line CCSu (RIL 92). The proportion of penetrated infection units that were stopped in association with a hypersensitivity reaction of the challenged epidermal cells ranged from 1% (Rec) to 11% (Vada) of the infection units. These results indicate that the QTLs that were mapped at the seedling stage should determine papilla-based, non-hypersensitive, basal or partial resistance.

33 Chapter 2

Candidate gene mapping Twenty three out of 39 EST-derived sequences representing candidate genes were mapped in at least one of the available mapping populations. Eighteen candidate genes were converted to CAPS markers. For one candidate gene, a SNP was used to design a dCAPS marker using the dCAPS Finder online software (http://helix.wustl.edu/dcaps/dcaps.html ). Two sequences were mapped as SCAR markers and two other sequences were previously mapped as RFLP markers. One sequence(ID: HO02M14) was polymorphic in the CCSu population but could not be assigned to a map position with confidence. Fiftheen candidate gene sequences remained for which no polymorphism was detected or for which it was not possible to obtain a clear PCR amplification product in any of the parental combinations (ID: HO14H18, HO14K08, HO16C19, HO26F22, HO13M13, HZ40O15, HI05K17, HO15M05, HO13K06, HW03O11, HU02G09, AJ344223, AJ518932, AJ518933, AM265370). The position of the mlo gene was determined directly in the LnVa map on chromosome 4H (Qi et al ., 1998; Shtaya et al ., 2006). Based on Collins et al ., (2001), Ror1 is located within a 0.1 cM distance of ABG452 marker on chromosome 1H. Ror2 was located on chromosome 5H based on its flanking markers as published by Collins et al ., (2003).

Co-localizations between candidate genes and QTLs The Chi-square test applied did not indicate a significant global co-localization between QTLs and candidate genes (Chi-square value, X2=0.79). Nevertheless, six out of 26 mapped candidate genes showed coincidence in their map positions with the QTLs identified for basal resistance to powdery mildew (Figure 2, Table 4). We defined coincidence of a QTL and a candidate gene, when the candidate gene mapped within the LOD-1 interval of the QTL in our integrated map. In this paper we only discuss those candidate genes which are within 5 cM from QTL peak markers because they are the most relevant ones. Despite similarities in resistance mechanism between basal resistance and mlo mediated resistance, none of the detected QTLs showed coincidence with the map positions of mlo and Ror2 genes. The peak marker of Rbgq6 , a QTL mapped in VaSu population at seedling stage, is located within a 0.1 cM distance of ABG452, a marker closely linked to Ror1 on chromosome 1H. Shtaya et al ., (2006) detected a QTL in LnVa population in the same region where Ror2 is present.

34 Which candidate genes are….

Co-localizations between QTLs and race-specific resistance genes In most cases, the map positions of powdery mildew resistance QTLs did not coincide with those of the race-specific resistance genes ( R genes). Only five out of 21 QTLs mapped in the same region where an R gene has been reported. We defined coincidence of a QTL and an R gene, when the putative map position of the R gene in our integrated map was located within the LOD-1 support interval of a QTL. Since the R-genes were mapped mostly in mapping populations that did not contribute to our integrated map, their map position in our integrated map is more indirectly obtained, and therefore the cM distance to our QTL peak marker is less precise than the map positions of the candidate gene-derived markers. A QTL mapped in LnVa and VaSu populations ( Rbgq9 ) showed coincidence with the known map position of the MlLa gene, a gene derived from “ Hordeum laevigatum ” and located on the long arm of chromosome 2H. This gene causes an intermediate type of hypersensitive reaction to avirulent isolates (Giese et al ., 1993; Marcel et al., 2007b). In both populations, the resistance QTL allele was contributed by Vada, a cultivar that carries the MlLa gene. Since the powdery mildew isolate used for evaluation of the populations at seedling stage was virulent to the MlLa carrying genotypes, it is plausible that the explained phenotypic variation in this region is due to a QTL and not to MlLa itself. One QTL detected on chromosome 7H in the OWBs ( Rbgq20 ) showed coincidence with the map position of mlt . A QTL mapped on chromosome 1H in NuTr population ( Rbgq4 ) showed coincidence with Mla and Mlk loci. Backes et al ., (2003) also reported co-localization of QTLs mapped in the cross Vada × 1B-87 on chromosomes 1H and 7H with the Mla and Mlf loci, respectively. A QTL mapped in the StMo population ( Rbgq5 ) on chromosome 1H showed coincidence with the map position of Mlnn gene. Finally, a QTL mapped in the NuTr population ( Rbgq12 ) on chromosome 4H showed coincidence with the map position of Mlg . But at the end, in the case of co-localizations between QTLs and R genes, only fine-mapping of the concerned loci can tell whether the phenotypic variation is due to the R gene or not.

35 Chapter 2

Table 4 . Characterization and map position of ESTs or cDNA-clone representing candidate genes for powdery mildew resistance in barley Clone Regul. 1 OEX or Function References Name of Position Co-localization ID/Acc. RNAi marker Chrom./cM with QTL(s) effect 2 HY05J20 up yes Bax-inhibitor 1 Hückelhoven et al ., 2003 GBR0504 6H/ 51.6 HO08H12 up yes HvGER4d Zimmermann et al ., 2006 WBE104 4H/ 135.8 Rbgq 13 HW05P16 up yes HvGER5a Zimmermann et al ., 2006 GBR0180 5H/ 97.4 HO06J21 up n.a. WIR1a Schweizer et al ., 1999 WBE101 7H/ 83.1 HO09K14 up n.a. WIR1b Zierold et al ., 2005 WBE100 5H/ 35.5 DQ647621 up yes HvGER3a Zimmermann et al ., 2006 WBE210 7H/ 88.6 HO02P09 up n.a. unknown Zierold et al. , 2005 WBE216 4H/ 108.5 HO07K06 up n.a. unknown Zierold et al ., 2005 WBE218 3H/ 149.0 HO11O12 up n.a. unknown Zierold et al ., 2005 WBE224 7H/ 23.0 HO13M20 up n.a. unknown Zierold et al ., 2005 WBE212 3H/ 11.8 Rbgq 10 HO14K22 up yes Receptor protein kinase-like Zierold et al ., 2005 WBE211 3H/ 142.7 HO14P02 up n.a. Receptor protein kinase-like Zierold et al ., 2005 WBE220 2H/ 136.2 HO12E03 up n.a. unknown Zierold et al ., 2005 WBE217 7H/ 33.0 HO07H15 up n.a. 4-Coumarate coenzyme A ligase Zierold et al ., 2005 WBE215 6H/ 59.8 Rbgq 18 HO40A01 up n.a. Hydrolase Zierold et al ., 2005 WBE214 4H/ 126.1 HO12F09 up yes SNAP-34 Douchkov et al ., 2005; Jensen et al ., 2007 WBE219 2H/ 63.6 Rbgq 8 HO06D23 up yes P-type ATPase Zierold et a l., 2005 WBE223 1H/ 79.8 HvPrx40 up yes Peroxidase Johrde et al ., 2008 WBE232 3H/ 81.2 L37358 up 3 n.a. 5 Lipoxygenase 2 Bachmann et al ., 2002 WBE228 5H/ 117.0 Rbgq 15 http://www.plexdb.org/index.php Hv07A14 up 3 n.a. 5 ACC synthase http://www.plexdb.org/index.php WBE229 2H/ 83.9 AJ536667 up yes WRKY1 transcription factor Eckey et al ., 2004 WBE230 7H/ 123.3 AJ534447 up 3 n.a. 5 Apoplastic invertase http://www.plexdb.org/index.php WBE231 2H/ 69.0 1 2 Regulation in barley leaves inoculated with Blumeria graminis ff.ssp.; RNAi effect, the effect of transient-induced gene silencing (TIGS) of test sequence on resistance phenotype 3 in the single-cell system ; OEX effect, the effect of single-cell transient over-expression of test sequence on resistance phenotype in the single-cell system; Regulation by Bgh or stem-rust inoculation in experiments BB2, BB4, BB7 or BB49 of Plant Expression Database (PlexDB) .Corresponding probe-set IDs: HV02H04, Contig12036_at; L37358, 4 5 HI02E21u_s_at; HY07A14, Contig15618_at; AJ534447, Contig11241_at.; Not significant; Not analyzed

36 Which candidate genes are….

Discussion Diversity of QTLs for powdery mildew resistance in barley Most mapping studies on quantitative, basal resistance, of barley to powdery mildew have been based on individual populations. Here we studied the genetic variation of basal resistance using multiple populations to assess also the diversity of the genes involved in the genetic variation. The QTLs conferring basal resistance were distributed over all seven chromosomes of barley without obvious clustering. In different mapping populations different sets of QTLs were involved in basal resistance. These indicated a great diversity for QTLs (genes) for this trait in barley. A similar great diversity has been reported in genes conferring basal resistance in some other barley-pathogen interactions, like barley-Puccinia hordei (Qi et al ., 2000), barley-Rhyncosporium secalis (Zhan et al ., 2008), barley-Fusarium (Fusarium head blight) (Sato et al ., 2008) and barley-spot blotch (Bilgic et al ., 2005). Qi et al ., (2000) reported, on the basis of two tested mapping populations, that many loci for basal resistance to leaf rust ( Puccinia hordei) should exist on the barley genome. Marcel et al ., (2007a) reported 19 QTLs contributing to the basal resistance of barley to leaf rust in five mapping populations. They showed that in each mapping population of barley different sets of QTLs segregate for basal resistance and only few QTLs were found to be in common in more than two different barley cultivars. QTL analysis for Fusarium head blight (FHB) resistance in different barley mapping populations indicate that quantitative resistance of barley to FHB is conditioned by many loci (Mesfin et al ., 2003). QTLs for powdery mildew resistance have been reported in different mapping populations of barley (e.g. Backes et al ., 1995; Backes et al ., 1996; Spaner et al ., 1998; Falak et al ., 1999; Backes et al ., 2003; Von Korff et al ., 2005; Shtaya et al ., 2006). We compared the position of the peak marker of the presently found QTLs with the peak markers of already reported QTLs using our high-density integrated map of barley. In four cases ( Rbgq2a, Rbgq4 , Rbgq7 and Rbgq8 ), the location of peak marker of QTLs are only about 0-2 cM away from previously reported QTLs for mildew resistance, and in two cases ( Rbgq2b and Rbgq14 ) the peak markers are 4-6 cM away. One of these QTLs ( Rbgq4 ) already has been reported in two different populations of barley, Blenheim × E224, Vada × 1B-87 (Thomas et al ., 1995; Backes et al ., 2003). This suggests that some QTLs found in our study might be the same as already reported QTLs in other genetic backgrounds.

Growth-stage specificity of QTLs for basal resistance Growth-stage dependent and independent QTLs both have been reported in many host-pathogen interactions, including powdery mildew and leaf rust on barley,

37 Chapter 2

septoria blotch on wheat, ascochyta blight on pea, and downy mildew on lettuce (e.g. Qi et al ., 1999; Backes et al ., 2003; Eriksen et al ., 2003; Prioul et al ., 2004; Marcel et al ., 2007a; Zhang et al ., 2009). In our study, only 4 out of a total of 21 QTLs were effective at both growth stages ( Rbgq2a, Rbgq7, Rbgq9, and Rbgq13 ), suggesting that QTLs for powdery mildew resistance are mainly growth-stage dependent. Similar results have been reported by Backes et al . (2003). They found that only one out of five QTLs that mapped in a cross between the barley cultivar ”Vada” and a wild barley (H. vulgare ssp. spontaneum line ”1B-87”) was growth-stage independent. This is also in agreement with the report of Mastebroek & Balkema-Boomstra (1991) in which it is concluded that the phenotypic expression of basal resistance of barley to powdery mildew is growth-stage dependent. Our histological observations confirm that the QTLs mapped in the present work contribute mainly to a prehaustorial non- hypersensitive basal resistance, rather than to an incomplete hypersensitive resistance.

Co-localization of candidate genes and R genes with QTL regions In field experiments under natural disease infection, R-gene resistance can behave as QTL resistance, since 1) the R-genes may have an intermediate effect (like MlLa ), and 2) the natural population of pathogen may consist of a mixture of virulent and avirulent races to a certain R-gene, resulting in a lower epidemic development on genotypes carrying the R-gene than in genotypes carrying the susceptibility allele (Parlevliet, 1983; Parlevliet & van Ommeren, 1985). Furthermore, QTLs that co- localize with R-genes for resistance may be allelic variants of R-genes or residual effect of defeated R-genes (Kelly & Vallejo, 2005). The molecular basis of quantitative (basal) resistance, like other genetically complex traits, is poorly understood. Candidate gene analysis can be considered as an important approach toward understanding the molecular basis of quantitative resistance, partly thanks to the availability of huge numbers of ESTs in databases. In the present study, we found a co-localization of QTLs for resistance with genes that have been suggested by reverse genetics to be involved in barley-powdery mildew interaction. Six out of 26 mapped candidate genes showed coincidence with the position of QTLs for powdery mildew resistance (Figure 2). Transient-induced gene silencing (TIGS) experiments showed that a t-SNARE protein, HvSNAP34, is involved in three types of durable, non-race specific resistances, viz. mlo -mediated, non-host and basal resistance (Douchkov et al ., 2005). Map co-localization of HvSNAP34 with a QTL ( Rbgq8) for powdery mildew resistance provides further evidence of the role of this gene in basal resistance. An other QTL ( Rbgq7) co-localized with the known map

38 Which candidate genes are….

position of Ror1 on chromosome 1H. None of the QTLs co-localized with mlo and Ror 2 genes. We conclude that, despite the similarity in resistance mechanism, there is little evidence that basal resistance is determined by allelic variants of the Mlo or Ror 2 genes. Map co-localization between resistance gene analogs (RGA) (see Glossary) and defense-related (DR) genes with QTLs for seedling and/or adult plant resistance have been reported in many other host-pathogen systems (Faris et al .1999; Geffroy et al ., 2000; Wang et al ., 2001; Prioul-Gervais et al ., 2007; Marcel et al ., 2007a). Hu et al ., (2008) established a candidate gene strategy that integrates linkage map, expression profile and functional complementation analyses with the purpose of cloning QTLs that are responsible for quantitative resistance of rice to bacterial blight and fungal blast. Using more than one mapping population, however, provides an impression on the diversity of the genes responsible for basal resistance. With a larger number of mapping populations and QTLs detected, it is possible to test a larger sample of QTLs for their statistical coincidence with the candidate genes. The use of an integrated map to compare data obtained in different populations also increases the chance of discovering relevant co-localizations. Here we prioritized the candidate genes by accumulating evidence of position, biological function and functional proof by TIGS. However, map co-localization of candidate genes with the QTLs involved in the trait variation is not a sufficient proof of causal relationship, and actual involvement of the candidate genes in the trait variation should be validated by complementation tests through genetic transformation and/or physiological analyses of genes at the mRNA and/or the protein level and by ultra-fine-mapping of both the candidatee gene and the QTL (Pflieger et al ., 2001). The integrated candidate gene strategy has proven to be a fast and efficient approach for isolation of genes with small effect (Hu et al ., 2008). Information gained from this approach may help breeders to use more efficiently the valuable sources of diversity to improve the level of basal resistance in crop plants.

Acknowledgements Reza Aghnoum was supported by the Agricultural Research and Education Organization (AREO) and the Ministry of Science Research and Technology of Iran. The excellent technical assistance of Anton Vels in greenhouse and field experiments is greatly appreciated. We are indebted to Dr. Ralph Hückelhoven (Technical University of Munich, Germany) for sharing five sequences of candidate genes; to Christine Boyd and Dr. Andris Kleinhofs from the Washington State University for making available the segregation data of many BIN markers; to Prof. Fred van Eeuwijk for allowing us to collect powdery mildew data from his field experiments of the NuTr population; to Dr. Rajeev K. Varshney from ICRISAT for providing primer aliquots and markers information on the new set of EST- SSR markers. Those EST-SSR markers have been developed at IPK in Germany. Marc-Antoine

39 Chapter 2

Bonvoisin from ESITPA in France is acknowledged for his hard work on mapping this new set of EST- SSR markers. We gratefully acknowledge the critical reading of the manuscript by Prof. Richard Visser.

References

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41 Chapter 2

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45 Chapter 2

46 Chapter 3

Transgressive segregation for extreme low and high level of basal resistance to powdery mildew in barley

R. Aghnoum, R. Tadayonirad and R.E. Niks

54 Chapter 3

Abstract Basal resistance of barley to powdery mildew is a quantitatively inherited trait that limits the growth and sporulation of barley powdery mildew pathogen by a non- hypersensitive mechanism of defense. Two experimental barley lines were developed with extremely high (ErBgh) and low (EsBgh) level of basal resistance to powdery mildew by convergent crossing followed by phenotypic selection between the most resistant and the most susceptible lines from four mapping populations of barley. We tried to verify the alleles of the seven identified QTLs in the four mapping populations from which the most resistant and the most susceptible individuals had been derived. Only four out of seven QTLs were confirmed to be present in the selected resistant lines. Surprisingly, none of the expected QTLs could be detected in the resistant line ErBgh. Our results showed that phenotypic selection in convergent crossing is very effective and resulted in very contrasting phenotypes for basal resistance. Histological observations showed that in ErBgh almost 90% of infection units failed to penetrate the epidermal cells in association with papilla formation. Established infection units may fail in formation of a substantial proportion of the secondary and subsequent haustoria. The extremely resistant and susceptible lines developed here are valuable material to be used in further experiments to characterize the molecular basis of basal resistance to powdery mildew.

Introduction R-gene triggered resistance is considered easy to select for, because of the large phenotypic effect. Most R-gene resistance appears to be associated with HR cell death (Watanabe & Lam, 2005). This resistance is only able to protect the plants against those races of the pathogen that possess an avirulence (Avr) gene corresponding to the R-gene. Pathogens like Blumeria graminis , the cereal powdery mildew pathogen, that possess a mixed (sexual and asexual) reproduction system and a high potential for gene flow due to the long-range wind dispersal of spores, are easily able to break down R-gene mediated resistance. Use of quantitative resistance has been proposed as a breeding strategy for durable protection against such a pathogens (McDonald & Linde, 2002). The minor gene, quantitative, resistance is often considered much more difficult to select for, due to the small contribution of each gene to the total level of resistance. Marker assisted selection has been proposed as a strategy to accumulate such resistance genes, in order to achieve high levels of this durable, non-race specific resistance (Castro et al ., 2003, Friedt & Ordon, 2007). Quantitative resistance of barley to powdery mildew is based on impaired haustorium formation in the attacked epidermal cells, and is not associated

48 Transgressive segregation for ….

with a hypersensitive reaction of challenged cells.This resistance also called basal resistance (see Glossary). Such a quantitative pre-haustorial resistance also occurs in various other mildew and rust fungal pathosystems (e.g. Carver, 1986; Niks, 1986; Asher & Thomas, 1987; Sillero & Rubiales, 2002). It has been reported frequently that among the progenies of a cross between two plant genotypes with intermediate levels of resistance or even susceptibility, descendants can be found with a higher or lower level of resistance compared to that of both parents (e.g. Jones, 1983; Wallwork & Johnson, 1984; Roderick & Jones, 1991). This phenomenon is known as transgressive segregation. Transgressive segregation implies that both parents carry a similar gene dose for the trait of interest, but those genes are located at different loci, and alleles for resistance from both parents can end up together in some of the progeny plants. Four barley mapping populations that had not been developed with the aim to study quantitative resistance to powdery mildew demonstrated that there is a great genetic diversity of minor genes for powdery mildew resistance, with very few of such genes in common between the populations (Chapter 2). Such a high diversity leads to substantial transgressive segregation for resistance levels. We presume that further accumulation of minor genes by convergent crossing and rounds of selection should result in even higher levels of resistance to be achieved. The objective of the present study was to investigate genetic plasticity of a limited, medium resistant barley gene pool for obtaining extremely susceptible and extremely resistant progeny by phenotypic selection for low and high resistance levels, respectively. On the basis of the knowledge of the contributing QTLs (see Chapter 2) we intended to test whether the resulting extremely susceptible and resistant progeny would indeed carry the expected QTL alleles for susceptibility or resistance.

Material and Methods Crossing and selection program In a previous study (Chapter2) we mapped QTLs for powdery mildew resistance at seedling stage in four mapping populations of barley, viz. one recombinant inbred line (RIL) population, L94 × Vada (LnVa) and three doubled haploid (DH) populations, Nure × Tremois (NuTr), Steptoe × Morex (StMo) and Oregon Wolfe Barleys (OWBs). Per mapping population, one line was selected with the lowest infection frequency on the primary leaf and the lowest haustorium formation rate of challenged epidermal cells. These homozygous most resistant lines were crossed pairwise (F 1), and their most resistant descendants (in F 2) were intercrossed again, forming a Double Cross

(DC), to develop, by phenotypic selection, a descendant (in DC-S1) with the highest

49 Chapter 3

level of quantitative (basal) resistance (Figure 1). In parallel, per mapping population, one line was selected with the highest infection frequency on the primary leaf and the highest haustorium formation rate of challenged epidermal cells, representing the most susceptible line of that mapping population. These lines with very low level of basal resistance were intercrossed in two rounds to form a Double Cross (DC) to accumulate QTL alleles for susceptibility. For development of DH barley lines with extremely high level of basal resistance, the DC-S1 seeds were allowed to self- pollinate and the most resistant DC-S2 plant was selected. A bulk of microspores of six plants (DC-S3) derived from the self-pollination of this DC-S2 plant was used for DHs production. For development of a DH barley line with extremely low level of basal resistance, three DC-S1 plants were used to make DH lines. From each DH plant three seedlings (12 days-old) were inoculated with powdery mildew and based on the infection frequency, four days after inoculation, two DH lines, one with the highest and one with the lowest infection frequency, were selected. The susceptible barley cv. Manchuria was used as reference.

Evaluation of basal resistance at seedling stage For evaluation of the mapping populations, selection of the most resistant and the most susceptible progenies and DHs, the following method was used. Barley plants were sown in boxes (37 × 39 cm) and were grown in spore free greenhouse compartments at 18-20 °C with 70% relative humidity and a photoperiod of 16 h. After 12 days, the fully expanded primary leaf was fixed in a horizontal position, with the adaxial side up. A settling tower was used for inoculation. Freshly produced conidia on young seedlings of the susceptible cultivar ‘Manchuria’ were blown into the tower. The density of inoculum was monitored by haemocytometer and was adjusted to 10- 15 conidia per mm 2. The inoculated plants were incubated in a growth chamber at 18- 20 oC with fluorescent light irradiance (102.8 micromol/m 2/s) supplied for 12 h per days. Four days after incubation, the barley accessions were scored by counting the number of young powdery mildew colonies per 2 cm 2 of leaf area using a frame with a 0.5 × 4 cm window and a magnifying glass (10x).

Histological assessment of infection Samples were collected from the middle part of inoculated leaves at 48 hai (hours after inoculation) and transferred to a solution of acetic acid-ethanol (3/1 v/v) to be cleared overnight. Then the leaf segments were stained with 0.1 mg ml-1 Trypan Blue in alcoholic lactophenol as described by Peterhänsel et al. (1997). To make the prepreations, the leaf segments were embedded adaxial side up in glycerol and

50 Transgressive segregation for ….

examined at 400× magnification using a transmitted white light Axiophot microscope (Zeiss, Germany). Only stomatal and interstomatal cells (type A and type B of epidermal cells following the terminology of Koga et al ., 1990) were examined. Long epidermal cells over-lying vascular bundles (type C) and subsidiary cells were not considered. Only those epidermal cells that were attacked by a single germinated conidium were evaluated.

Verification of QTLs via putative flanking markers One DH line was selected from each double cross DH population. For the resistant selection (DC-R) there were 132 DHs, for the susceptible selection (DC-S) 129 DHs. From DC-R and DC-S the most resistant and the most susceptible DH lines, respectively, were selected based on the phenotypic reaction of DHs to powdery mildew. The inoculation with powdery mildew and evaluation was performed in the same way as described earlier. The selected lines were genotyped for flanking markers of the seven identified QTLs (Table1).

Results Development of barley lines with extremely low and high level of basal resistance to powdery mildew

Based on both macroscopic and histological data, DC-S1 families showed transgressive segregation both towards resistance and susceptibility (data not shown). One DH line was selected from each double cross (DC-R and DC-S) combination out of 132 and 129 total DH lines produced, respectively. The extremely resistant and the extremely susceptible barley lines to Bgh were called ErBgh and EsBgh, respectively. ErBgh and EsBgh showed a remarkable difference in infection frequency of powdery mildew at 6 dai (Figures 2 and 3). Histological evaluations revealed that in the most resistant lines of the mapping populations always a higher proportion of germlings was stopped in association with papilla formation at the sites of attempted penetration than in the most susceptible lines (Figure 4). The ErBgh line allowed only about 12 % and the EsBgh line 67% of the mildew germlings to form a primary haustorium (Figure 4). In ErBgh a considerably lower proportion of secondary penetration attempts along the elongated secondary hyphae (ESH) resulted in a haustorium than in EsBgh at 48 hai (19% and 60% respectively). Interestingly, in ErBgh, most of the successful secondary penetrations were observed in the same cell as where the first haustorium was formed (Figure 5). On ErBgh no substantial hypersensitivity reaction was observed.

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Our results demonstrated that just by visual selection QTLs from different sources can be combined to reach an extreme level of basal resistance.

QTL verification We tried to verify the alleles of the seven identified QTLs in the four mapping populations from which the most resistant and the most susceptible individuals had been derived. It seemed reasonable to presume that line ErBgh would combine for all or most of the QTLs the resistance allele, EsBgh for all or most of the QTLs the susceptibility allele. We checked the genotype of the most resistant and the most susceptible genotypes of each mapping population that were used for making the first round of crossings (Table 1). Only four QTLs ( Rbgq14, Rbgq2a, Rbgq19 and Rbgq10 ) were confirmed to have the resistance allele in those four selected resistant lines (Table 1). Surprisingly, none of the QTLs for which we can have a conclusion (Rbgq14, Rbgq2a, Rphq19 and Rphq10 ) could be detected in the resistant line ErBgh. Only three QTLs ( Rbgq2a, Rbgq19 and Rbgq15 ) were confirmed to have the susceptibility allele in the selected susceptible lines. Surprisingly, none of the QTLs for which we can have a conclusion ( Rbgq2a, Rbgq19 and Rbgq15 ) could be detected in the susceptible line EsBgh. The verification of the QTL alleles present in ErBgh and EsBgh was hampered by at least two technical points. 1-Lack of multiple allelic variations makes a QTL peak or flanking marker-allele not unique for selection if this allele also occurs in at least one of the parents of the other mapping populations. This is especially true for markers like AFLP, where each marker has only two variants (“0” or “1”). 2 -We searched on the latest integrated barley linkage map (Chapter 2) for multiple allelic SSR markers close to the target QTLs, to increase the chance that the allele that was indicative for a particular QTL allele, did not occur in any of the other ancestral lines. However, we found such SSR markers often at some distance from the peak marker of the QTL. Therefore, recombination between the marker locus and the QTL might occur during the crossing procedure. In case of such a distance, absence of the indicative marker allele does not prove absence of the QTL allele.

52 Transgressive segregation for ….

Mapping QTLs for powdery mildew Selection of the most resistant line of resistance at seedling stage in four each population mapping populations

Figure 1. A schematic representation of a crossing programs to develop a Two cycles of recurrent selection to barley line with extreme high level of accumulate QTLs for resistance basal resistance to powdery mildew. A R1 × R 2 R3 × R 4 parallel crossing program followed to develop a line with extreme low level of resistance. F1 Selfing F1

F2 Selection F 2

F2 R1 × R2 × F2 R3 × R4

DC-S1

DC-S2 Selection

DC-S3

Making DH lines

Figure 2. Phenotypeof the extremely resistant (ErBgh, left) and the Selection of the extremely resistant extremely susceptible (EsBgh) barley line lines six days after inoculation with Bgh with the same inoculom density and incubation conditions.

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Table1: Characteristics of QTLs mapped at seedling stage for powdery mildew resistance and the genotype of the most resistance and the susceptible lines used for crossing Population QTLs Peak marker LOD Exp% a Donor b R parent c S parent d

OWBs Rbgq20 ABG704 3.5 18.3 Rec - + Rbgq14 Bmac0047e 4.9 19.9 Dom + +

NuTr Rbgq2a bPb-0312 6.1 24.8 Tremois + - Rbgq19 scssr05599 5.55 22.4 Nure + -

LnVa Rbgq2b E35M48-410a 2.8 19.8 L94 - + Rbgq15 E33M58-306 3.1 21.5 Vada - -

StMo Rbgq10 MWG571C 4.7 15.5 Morex + +

a The proportion of the phenotypic variance explained by the QTL b Donor of the resistance allele c The genotype of the most resistant (R ) line of each population regarding the expected QTL; + indicates that the line contained a molecular marker that suggested the expected resistance allele of the QTL, - that the line contained a molecular marker that suggested the unexpected susceptibility allele. d The genotype of the most susceptible (S) line of each population regarding the expected QTL; + indicates that the line contained a molecular marker that suggested the expected susceptibility allele of the QTL, - that the line contained a molecular marker that suggested the unexpected resistance allele.

54 Transgressive segregation for ….

90 80 70 60 50 40 30

Infection frequency Infection 20 10 0

-r -r -r -s s -s h o r g ria Va Bs Tr-r Va u WBs- EsB ch StM Ln Nu StMo-s Ln NuT ErBgh n OW O a M Figure 3. Infection frequency [colonies per 2 cm 2] of the most resistant (r) and the most susceptible (s) genotypes of different mapping populations. LnVa (L94 × Vada), StMo (Steptoe × Morex), OWBs (Oregon Wolf Barleys) and NuTr (Nure × Tremois). ErBgh represents the extremely resistant and EsBgh the extremely susceptible DH barley line to Bgh derived from the double crosses. Data were collected at 4 dai.

80 70 60 50 40 30 20

Penetration efficiency% 10 0

r r s s h -r - - h g Va-r Tr- Tr B tMo- tMo WBs S WBs-s ErBg Es churia S Ln O Nu LnVa-s O Nu n Ma Figure 4. Penetration efficiency (% successfully penetrating germlings) of Bgh on the most resistant (r) and the most susceptible (s) genotypes of different mapping populations. ErBgh represents the extremely resistant and EsBgh the extremely susceptible DH barley line to Bgh derived from the double crosses.

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Figure 5. Develpoment of Bgh (48 hai) on ErBgh. A, a successful primary penetration resulted in a haustorium and a failed secondary attempted penetration (SAP). B, a closer view of failed SAP showed in fig. A. C, a successful primary penetration resulted in formation of a secondary haustorium in the same cell. The scale bar represents 100 m.

Discussion Accumulation of genes with minor effect by phenotypic selection Our data support the proposed strategy that genotypes with much higher (and lower) level of resistance than the parents can be selected from progenies derived from convergent crosses between several parents with intermediate levels of resistance, due to high transgression of the genetic components from both parents (Wallwork & Johnson, 1984). This high success of accumulation of quantitative genes for basal resistance can be ascribed to the great diversity of such genes in the barley germplasm, as shown in Chapter 2, involving different loci in each parental pair. Such an accumulation was, for the same reason, also easily achieved by Parlevliet & Kuiper (1985), in barley to barley leaf rust ( Puccinia hordei ). Also to that pathogen loci for basal resistance occur all over the barley genome (Qi et al ., 2000; Marcel et al ., 2007), making transgression for basal resistance a very common phenomenon.

Efficiency of phenotypic selection to achieve a high level of basal resistance Our results showed that phenotypic selection in convergent crossing is very effective and resulted in very contrasting phenotypes for basal resistance. MAS has some problems, like the lack of unique markers close to the peak marker of the QTLs of

56 Transgressive segregation for ….

interest, especially if more than two parents will be used in a convergent crossing programme. With strong enough linkage disequilibrium (see Glossary) between markers and QTLs (Dekkers & Hospital, 2002), MAS should in theory be easier, faster, cheaper, or more efficient than classical (phenotypic) selection but in practice the MAS is not always as efficient as expected (Hospital, 2008). It should be pointed out here that the efficiency of MAS also depends on the type of the trait and to the population. Phenotypic selection is more effective than MAS in some cases or at least both are of the same value (Romagosa et al ., 1999; Flint-Garcia et al ., 2003). Flint- Garcia et al . (2003) compared the efficiency of phenotypic selection versus Marker- assisted selection for rind penetrometer resistance (RPR) and European corn borer (ECB). They reported that MAS for high and low RPR was effective in the three populations studied. Phenotypic selection for both high and low RPR was more effective than MAS in two of the populations. However, in a third population, MAS for high RPR using QTL effects from the same population was more effective than phenotypic selection. MAS is probably not needed for a rather simple trait like quantitative powdery mildew resistance. Selection for powdery mildew resistance is quite straightforward, plenty of genes in the ancestors are available that can be accumulated and non-genetic variation is relatively low if inoculum is applied homogeneously over the plants to be compared. MAS may be more useful to introgress or accumulate QTLs for such traits like drought and salt tolerance that are difficult to select for phenotypically. However, also in such cases unique marker alleles are required if a convergent crossing scheme is used. It is remarkable that our initially selected most resistant and most susceptible lines Failed SAP from the mapping populations did not contain (nearly) all the expected QTL alleles (Table 1). One possible explanation may be that the effect of QTLs depends strongly on the genetic background. Marcel et al. (2008) showed that some QTLs for basal resistance to P. hordei had a particularly high effect if combined with other QTLs, that alone had hardly any effect. Such interactions would make phenotypic selection probably more successful than MAS, in case the partner QTLs of such interactions are still unknown.

High level of basal penetration resistance in ErBgh line Basal resistance of barley to powdery mildew is mainly based on impairment of haustorium formation. The observation that established infection units in ErBgh may fail in formation of a substantial proportion of the secondary and subsequent haustoria (Figures 5A and B) is unexpected. In other barley and oat genotypes (Carver & Carr, 1978; Gustafsson & Claesson, 1988), lower secondary haustorium

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formation is reported but it is not mentioned whether this is due to failure of attempted secondary penetration or to slow growth resulting in fewer penetration attempts. The percentage of successful primary penetration in ErBgh is nearly as low (12%) as the lowest (10%) reported ever (Carver, 1986), excluding non-host and mlo resistance. It seems plausable that further rounds of convergent crossing and selection, involving parents with high quantitative resistance might easily increase resistance levels even further. It should be further investigated whether the failed secondary and attempted penetration in ErBgh is due to papilla formation or that other mechanisms are involved. The present observation that in ErBgh at 48 hai the second and next haustoria are often formed in the same cell as the first haustorium (Figure 5C) is intriguing. A similar phenomenon was observed in SusBgt lines that show a high level of susceptibility to the non-adapted wheat powdery mildew, B. graminis f.sp. tritici (Chapter 4). This is a further indication that basal host resistance has similarities with non-host resistance. We consider the extremely susceptible and resistant lines developed here as valuable material to be used in further experiments to characterize the molecular basis of basal resistance to powdery mildew. Additional work on this material could also lead to a critical comparison of phenotypic versus marker assisted selection for a quantitative trait like basal resistance.

Acknowledgements The excellent technical assistance of Anton Vels in greenhouse experiments is greatly appreciated. Dr. TC. Marcel is acknowledged for his help in QTL verification using SSR markers.

Reference Asher MJC, Thomas CE. 1987. The inheritance of mechanisms of partial resistance to Erysiphe graminis in spring barley. Plant Pathology 36: 66-72. Carver TLW, Carr AJH. 1978. Effects of host resistance on the development of haustoria and colonies of oat mildew. Annals of Appliled Biology 88: 171–178. Carver TLW. 1986. Histology of infection by Erysiphe graminis f. sp. hordei in spring barley lines with various levels of partial resistance. Plant Pathology 35: 232–240. Castro AJ, Chen X, Hayes PM, Johnson M. 2003. Pyramiding quantitative trait locus (QTL) alleles determining resistance to barley stripe rust: Effects on resistance at the seedling stage. Crop Science 43: 651-659. Dekkers JCM, Hospital F.2002. The use of molecular genetics in the improvement of agricultural populations. Nature Reviews Genetics 3: 22–32. Flint-Garcia SA, Darrah L, McMullen M. 2003. Phenotypic versus marker-assisted selection for stalk strength and second-generation European corn borer resistance in maize. Theoretical and Applied Genetics 107: 1331–1336.

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Friedt W, Ordon F. 2007. Molecular Markers for Gene Pyramiding and Disease Resistance Breeding in Barley. In:Varshney RK, Tuberosa R, eds, Genomics-Assisted Crop Improvement:Vol. 2: Genomics Applications in Crops, 81–101. Gustafsson M, Claesson L. 1988. Resistance to powdery mildew in wild species of barley. Hereditas 108: 231–237. Hospital F. 2008. Challenges for effective marker-assisted selection in plants. Genetica 10.1007/s10709-008-9307-1 Jones IT. 1983. Transgressive segregation for enhanced level of adult plant resistance to mildew in the oat cross Mostyn x Maldwyn. Euphytica 32: 499-503. Koga H, Bushnell WR, Zeyen RJ. 1990. Specificity of cell type and timing of events associated with papilla formation and the hypersensitive reaction in leaves of Hordeum vulgare attacked by Erysiphe graminis f. sp. hordei . Canadian Journal of Botany 68: 2344–2352. Marcel TC, Gorguet B, Ta MT, Kohutova Z, Vels A, Niks RE. 2008. Isolate specificity of quantitative trait loci for partial resistance of barley to Puccinia hordei confirmed in mapping populations and near-isogenic lines. New Phytologist 177: 743–755. Marcel TC, Varshney RK, Barbieri M, Jafary H, de Kock MJD, Graner A, Niks RE. 2007. A high- density consensus map of barley to compare the distribution of QTLs for partial resistance to Puccinia hordei and of defence gene homologues. Theoretical and Applied Genetics 114: 487– 500. McDonald BA, Linde C. 2002. The population genetics of plant pathogens and breeding strategies for durable resistance. Euphytic a 124: 163-180. Niks RE. 1986. Failure of haustorial development as a factor in slow growth and development of Puccinia hordei in partially resistant barley seedlings. Physiological and Molecular Plant Pathology 28: 309–322. Parlevliet JE, Kuiper HJ. 1985. Accumulating polygenes for partial resistance to barley leaf rust, Puccinia hordei . 1. Selection for increased latent period. Euphytica 37: 7–13. Peterhänsel C, Freialdenhoven A, Kurth J, Kolsch R, Schulze-Lefert P. 1997. Interaction analyses of genes required for resistance responses to powdery mildew in barley reveal distinct pathways leading to leaf cell death. Plant Cell 9: 1397–1409. Qi X, Fufa F, Sijtsma D, Niks RE, Lindhout P, Stam P. 2000. The evidence for abundance of QTLs for partial resistance to Puccinia hordei on the barley genome. Molecular Breeding 6: 1–9. Roderick HW, Jones IT. 1991. The evaluation of adult plant resistance to powdery mildew ( Erysiphe graminis f.sp . avenae ) in transgressive lines of oats. Euphytica 53: 143-149. Romagosa I, Han F, Ullrich SE, Hayes PM, Wesenberg DM. 1999. Verification of yield QTL through realized molecular marker-assisted selection responses in a barley cross. Molecular Breeding 5: 143–152. Sillero JC, Rubiales D. 2002. Histological characterization of the resistance of faba bean to faba bean rust. Phytopathology 92: 294–299. Wallwork H, Johnson R. 1984. Transgressive segregation for resistance to yellow rust in wheat. Euphytica 33: 123–132. Watanabe N, Lam E. 2005. The Hypersensitive Response in Plant Disease Resistance. In: S, Tuzun E, Bent eds, Multigenic and Induced Systemic Resistance in Plants. Springer. 83-111.

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54 New perspectives to identify genes that…

New perspectives to identify genes that determine non-host resistance of barley to non-adapted Blumeria graminis forms

R. Aghnoum and R.E. Niks

Submitted

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Abstract The genetic basis of non-host resistance of barley to non-adapted formae speciales of Blumeria graminis is not known, since there is no barley line that is susceptible to, for example, the wheat powdery mildew pathogen, Blumeria graminis f.sp. tritici ( Bgt ). We identified barley accessions with rudimentary susceptibility to the non-adapted Bgt . Those accessions were intercrossed in two cycles and selected two lines, called

SusBgt SC and SusBgt DC , with exceptional susceptibility to Bgt at seedling stage.The quantitative variation among barley accessions and in the progenies after convergent crossing suggests a polygenic basis for this non-host resistance. Both lines show a remarkable level of haustorium formation and colony development by the non- adapted target mildew. The SusBgt lines and their ancestor lines also allowed haustorium formation and conidiation by four out of seven tested non-adapted Blumeria forms. Component analysis of the infection process suggested that non-host resistance factors are specific to the form and developmental stage of Blumeria . Resistance to establishment (first haustorium), colonization (subsequent haustoria) and conidiation are not clearly associated. The lines developed will serve to elucidate the genetic basis of non-host resistance in barley to Bgt , and they are useful tools in gene expression and complementation studies on non-host resistance.

Introduction The majority of plant species are immune to the majority of potentially pathogenic microbial invaders. This is the most common and broadest spectrum resistance, termed non-host resistance. It is defined as resistance shown by all members of a plant species against all members of a given pathogen species (Heath, 2000; Thordal-Christensen, 2003). Non-host resistance relies on a variety of preformed and inducible mechanisms of defense (Thordal-Christensen, 2003; Mysore & Ryu, 2004). Pathogens that are able to overcome the preformed defense mechanisms encounter extracellular surface receptors that recognize general elicitors, the so-called pathogen-associated molecular patterns (PAMP) (see Glossary) (Nürnberger et al ., 2004; Chisholm et al ., 2006). The pathogen should suppress the PAMP-triggered immunity (PTI) in order to establish a compatible interaction and successfully colonize the plant (Chisholm et al ., 2006). Those pathogen species that are not able to suppress the PTI are often called non-adapted, inappropriate or heterologous pathogens. It is presumed that would-be pathogens deliver effectors (see Glossary) in the attacked plant cells that may suppress PTI if there is sufficient correspondence between effectors and their respective operative targets (Block et al., 2008; van der

62 New perspectives to identify genes that…

Hoorn & Kamoun, 2008). The degree of correspondence between effectors and targets may determine the host status of a plant to a would-be pathogen (Niks & Marcel, 2009). Two interesting questions are whether different stages of development of a pathogen in a plant may require different sets of effectors and whether different strains of a pathogen species need to address the same operative targets in a particular plant species. Powdery mildew in cereals is caused by the biotrophic Ascomycete fungus Blumeria graminis (Bg ) formerly known as Erysiphe graminis. Bg has a high level of biological specialization for its cereal and grass hosts. Barley is considered a strict non-host plant species to the wheat forma specialis, Blumeria graminis f.sp. tritici ( Bgt ), and also to the forms of Blumeria graminis that are pathogenic to oats and rye (Hardison, 1944; Eshed & Wahl , 1970; Menzies & MacNeill, 1989). At the macroscopic level, no barley accession has been identified that allowed Bgt colonies to develop (Menzies & MacNeill, 1989; Olesen et al ., 2003; Atienza et al. , 2004). At the microscopic level, however, some barley accessions have been reported to permit some development of Bgt (Tosa & Shishiyama, 1984; Trujillo et al ., 2004). Penetration of the plant epidermal cell walls and subsequent haustorium formation, hereafter called establishment, is a critical stage in the infection process. In compatible interactions of barley with the adapted powdery mildew pathogen, Blumeria graminis f.sp. hordei (Bgh), the fungus can successfully penetrate through epidermal cell walls and form haustoria to obtain nutrients from the host cells. In non-host interactions of barley with non-adapted formae speciales such as Bgt , establishment typically fails in association with formation of cell wall appositions (papillae), in some cases supplemented with the hypersensitive cell death reaction (HR) of single attacked cells. In the interaction of barley with the adapted powdery mildew, Bgh, a proportion of attempted germlings are also stopped by such defense mechanisms (Tosa & Shishiyama, 1984; Hückelhoven et al ., 2001; Trujillo et al ., 2004). It is unknown which genetic factors determine that barley is a host to Bgh but a non- host to non-adapted formae speciales of Bg . Since there is no barley line that is susceptible to, for example, Bgt , inheritance studies are not possible. Some candidate genes have been implicated to play a role in resistance of barley to the wheat forma specialis (Bgt ) by transient overexpression or transient-induced gene silencing (TIGS) in single epidermal cells (Miklis et al ., 2007; Douchkov et al ., 2005; Eichmann et al ., 2004; Schweizer, 2007), but it is not known whether allelic variation between these genes explains the host status difference between wheat and barley to Bgt . Barley is indeed a model plant in which to study the molecular basis of basal and non-host resistance to powdery mildews (Hückelhoven, 2007; Schweizer , 2007; Collinge et al. ,

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2008). We set out to identify the genes that are responsible for natural variation in host status and non-host resistance level. In the present paper, we identified barley accessions with rudimentary susceptibility to the non-adapted wheat powdery mildew ( Bgt) and crossed them to develop experimental barley lines with exceptional susceptibility to Bgt . We quantified components of infection development of Bgh, Bgt and several other non-adapted powdery mildew formae speciales and species on these experimental lines to determine the specificity of resistance factors.

Materials and Methods Plant and Pathogen Materials A collection of 439 accessions of barley was evaluated at the seedling stage to the non-adapted wheat powdery mildew fungus, Bgt . The collection consisted of 136 accessions of Hordeum spontaneum and 303 accessions of Hordeum vulgare : viz ., 227 accessions (landraces and wild H. spontaneum barley from the ICARDA barley germplasm collection) provided by Dr. Rajeev K. Varshney, 54 accessions received from the Centre for Genetic Resources (CGN), Wageningen, the Netherlands and 22 parental lines of available mapping populations of barley at the Laboratory of Plant Breeding, Wageningen University. The seeds of barley cv. Turkey 290 were kindly provided by Dr. H.E. Bockelman (National Small Grains Collection, USDA, USA). An isolate of Bgt (Swiss field isolate FAL92315), kindly provided by Dr. Patrick Schweizer (IPK, Germany), was propagated on the susceptible wheat cv. Vivant and used for inoculation. The lines with some level of susceptibility to Bgt were intercrossed in two cycles and resulted in two lines with exceptional susceptibility to Bgt (see Results section). These lines were named SusBgt SC and SusBgt DC . SusBgt lines and the parental lines were tested to several non-adapted powdery mildew formae speciales (ff. spp.) and species. The isolates will be referred to according to their source host. The following non-adapted ff. spp. of Bg were tested: Blumeria graminis f.sp. avenae (Bga ), the powdery mildew of oat ( Avena sativa ); Blumeria graminis f.sp . secalis (Bgs ), the powdery mildew of rye ( Secale cereale ); and four isolates collected from wild grasses, Hordeum murinum, Blumeria graminis f.sp . hordei-murini (Bghm ); Agropyron repens, Blumeria graminis f.sp. agropyri ( Bgar ); Bromus mollis , Blumeria graminis f.sp. bromi ( Bgb ); and Dactylis glomerata, Blumeria graminis f.sp . dactylidis (Bgd ). All of these isolates were collected in the Wageningen region and maintained in isolation on their respective host plants. We also tested two non-adapted species, viz. Oidium neolycopersici , the powdery mildew of tomato, and Sphaerotheca fuliginea , the powdery mildew of cucumber.

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Inoculation Three seedlings of each barley accession were grown in boxes 37 × 39 cm in size. The susceptible wheat cv. Vivant was sown in each box to monitor the effectiveness of inoculation. Turkey 290, SusPmur and SusPtrit which have been reported to allow relatively high haustorium formation by Bgt (Tosa & Shishiyama, 1984; Trujillo et al ., 2004) were included as references. The fully expanded primary leaves of 12-day-old seedlings were stapled horizontally, adaxial side up. To make a dense inoculation, freshly produced conidia of a Bgt isolate formed on the susceptible wheat cv. Vivant were blown onto the leaves to reach a density of approximately 50 conidia per mm 2. The inoculated plants were incubated in a growth chamber at 18-20 °C with 70% relative humidity and a photoperiod of 16 h (200 micromol/m 2/s).

Macroscopic and microscopic evaluation of barley-Bgt interaction First, macroscopic evaluations of all tested accessions were carried out using a magnifying glass (10x) eight days after inoculation (dai). Those accessions that macroscopically showed some degree of susceptibility were selected. These accessions were sown and inoculated again for quantification of haustorium formation and conidiation under the microscope. At two time points, 72 hours after inoculation (hai) and 8 dai, a leaf segment approximately 3 cm long was collected from the middle part of the infected primary leaves. Leaf segments were transferred to a solution of acetic acid-ethanol (3:1 v:v) to be cleared for at least 3 hours. Then, the leaf segments were stained in Coomassie Brilliant Blue according to the method of Wolf & Fric (1981). The experiment was carried out in two consecutive replications. Per barley accession, 200 observed germlings were scored for the result of the first penetration attempt to establish a haustorium. Because different types of host epidermal cells show different degrees of resistance to penetration (Lin & Edwards, 1974; Koga et al ., 1990) and penetration attempts by different germlings on the same cell may induce resistance or susceptibility (Carver et al ., 1999; Olesen et al ., 2003), we considered only cells that were attacked by a single conidium and only epidermal cells adjacent to stomata and interstomatal epidermal cells (type A and type B of epidermal cells following the terminology of Koga et al ., 1990).

Evaluation of susceptibility of SusBgt lines SusBgt lines and the parental lines were tested to non-adapted powdery mildew isolates in order to quantify the degrees of haustorium formation, hypersensitive and non-hypersensitive mechanism of defense and conidiation. At 72 hai, leaf samples were cut and placed in a solution of 1 mg ml−1 3,3'-Diaminobenzidine (DAB)-HCL, pH

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3.8, which allows whole-cell accumulation of H 2O2, for 8 h and subsequently transferred to a solution of acetic acid-ethanol (3:1 v:v) to be cleared overnight. Then the leaf segments were stained with 0.1 mg ml-1 Trypan Blue in alcoholic lactophenol as described by Peterhänsel et al., (1997). H2O2 accumulation appears as a reddish- brown coloration of epidermal cells and indicates the degree of HR (Thordal- Christensen et al ., 1997).

Results Genotypic variation in interaction of barley with the wheat powdery mildew pathogen The majority of the 439 barley accessions were (nearly) immune, showing no macroscopic symptoms or signs of infection whatsoever with the non-adapted pathogen ( Bgt ) even 10 days after inoculation. However, a few barley accessions were identified that showed a low degree of susceptibility to Bgt . On these accessions very small lesions, or even in a few cases tiny colonies, were visible by 10x magnifying glass or by naked eye, indicating some haustorium formation by the non- adapted pathogen. Six such accessions were examined under the microscope to quantify the amount of haustorium formation in comparison with lines Turkey 290, SusPtrit and SusPmur and with the immune barley cv. Vada. The cellular reactions of these selected lines to Bgt is shown in Figure 1. On barley cv. Vada, all germlings were stopped at the penetration stage in association with the deposition of cell wall appositions (papillae), and the fungus could not develop further to establish any haustoria. On the other accessions, the large majority of germlings were also stopped at penetration stage, but 12 to 25 % of the germlings succeeded in penetrating through the epidermal cell wall and established a haustorium and elongated secondary hyphae (ESH). Accessions with similar rates of establishment by Bgt , such as Hsp17 and SusPtrit, could differ greatly in the rate of conidium formation (Figure 1). The results confirm earlier reports (Tosa & Shishiyama, 1984; Trujillo et. al ., 2004) that there are differences among barley accessions in the degree to which they allow the non-adapted wheat powdery mildew pathogen to establish haustoria. Such rare barley accessions with some level of susceptibility to this non-adapted mildew were used as parental lines to accumulate genes for susceptibility to Bgt .

66 New perspectives to identify genes that…

35 30 25 20 15 10

Interaction sites[%] 5 0

9 2 7 0 e 9 ur hi 2 trit m P Pm Vada azar ha HSP14 HSP17 Hiproly B akot rkey Sus us li C t u S u im T ris B T Figure 1. Penetration efficiency and conidiation of Blumeria graminis f.sp. tritici (Bgt ) on different barley accessions that showed some level of susceptibility to Bgt . The immune barley cv. Vada was used as a reference. White columns represent the successful primary penetration with a haustorium, collected from two leaf segments (each from independent inoculation experiments) fixed at 72 hai. In each leaf segment, 100 attempted penetrations were recorded. Gray columns represent the percentage of all successful primary penetrations that resulted in formation of conidia at 8 dai. Data represent the means of two leaf segments collected from two independent inoculation experiments.

Development of SusBgt lines, susceptible to the non-adapted wheat powdery mildew Based on macroscopic and microscopic observations, including the degree of conidiation, four accessions of barley, viz. Hiproly (CGN00588; Egypt), Trisuli Bazar 9 (CGN00931; Nepal), Chame 2 (CGN01042; Nepal) and SusPtrit (Atienza et al ., 2004) were selected as the most promising lines for crossing to accumulate genes for susceptibility to Bgt . We made pairwise crosses between these four lines (Trisuli Bazar 9 × Hiproly) and (SusPtrit × Chame 2) (Figure 2). The second generation offspring (F 2) derived from each of the two crossing combinations were evaluated first macroscopically for susceptibility to the Bgt isolate, and then a few F 2 plants from each crossing combination that seemed to be more susceptible than the parents were tested microscopically for penetration efficiency and conidiation. A double cross (DC) was made between a selected individual F 4 plant derived from the cross Trisuli Bazar

9 × Hiproly and a selected individual F 3 plant derived from the cross SusPtrit ×

Chame 2. Four DC-S1 plants coming from the same single DC plant were used for making doubled haploid (DH) lines by microspore culture. We had expected additional transgression in the DC (because it had four susceptible ancestors) compared to the single cross (SC) with only two ancestors. However, during the procedure, the progeny from the cross SusPtrit × Chame 2 seemed so susceptible that we produced also a DH from that lineage. Two selected F 4 individuals derived from one selected F 3 plant from the single cross (SC) SusPtrit × Chame 2 were also used for making DH

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lines. Progeny from selfed DH plants were first evaluated macroscopically, and those lines that showed a clear susceptibility were analyzed microscopically. Since the selected DC and SC lines looked similarly susceptible, we decided to test them both extensively. The most Bgt -susceptible lines derived from the single cross and double

cross were named SusBgt SC and SusBgt DC .

Fig ure 2 . Two cycles of convergent crossing in barley to accumulate genes for susceptibility to the wheat powdery mildew ( Blumeria graminis f.sp . tritici).

Crossing Selfing Selection of the Selfing Selfing Double cross most susceptible (selection) (selection) F2 plants

P 1 × P 2 F 1 F 2 F3 F4 F4 P1 × P2 × F 3 P3 × P4 P 3 × P 4 F 1 F2 F3

DC-S1

Making DH lines

P 1 = SusPtrit Selection of the most susceptible DH line

P2 = Chame 2 SusBgt SC Selection of the most

P 3 = Trisuli Bazar 9 susceptible DH line

SusBgt DC

P 4 = Hiproly

Susceptibility of SusBgt lines to the adapted ( Bgh ) and non-adapted wheat powdery mildew pathogen ( Bgt ) SusBgt lines and all the parental lines showed a compatible interaction (infection type 4 according to the scale of Mains & Dietz (1930)) with the barley powdery mildew. Interestingly, SusPtrit and both SusBgt lines arrested at least 50% of the infection units of the adapted pathogen, Bgh, from developing haustoria. This indicates a rather high level of basal resistance to Bgh (Figure 3) . SusBgt lines allowed the non-adapted Bgt to form many colonies that were clearly visible by the naked eye at 8 dai and even earlier (Figure 4A). In the barley accessions, a proportion (depending on genotype, 12 to 27%) of challenged epidermal cells without detectable haustoria responded by HR (Figure 5A).

68 New perspectives to identify genes that…

There were great differences between the accessions in the rate of cells in which a haustorium was formed. Vada was fully resistant to penetration by Bgt . Both SusBgt

SC and SusBgt DC lines showed a higher level of haustorium formation by Bgt than the parental lines (Figure 5A). In SusBgt SC and SusBgt DC , 51% and 59%, respectively, of germlings had succeeded in penetration and haustorium formation, while in Chame 2, the most susceptible parental line, only 35% of germlings had successfully formed a first haustorium. In SusBgt DC , which showed the highest frequency of successfully penetrated cells, a larger proportion of penetrated cells with a haustorium (59%) were stopped by single-cell HR than in SusBgt SC (42%) and the parental lines. The highest frequency of successfully penetrated epidermal cells without HR was detected in

SusBgt SC (29%).

100% 90% 80% 70% 60% 50% 40% 30% Interaction sites sites Interaction 20% 10% 0%

9 2 it r r C t D proly za ame Ba Bgt SC Bgt Hi i Ch SusP s ul Sus Su is Tr Figure 3. Defense reactions of SusBgt lines and their parental lines to the barley powdery mildew (Bgh ) at 48 hai. White columns represent failed primary penetration. Black columns represent the successful primary penetration with a haustorium and with hypersensitivity (HR). Cross-hatched columns represent the successful primary penetration with a haustorium but without HR. Data represent the means of four leaf segments collected from two independent inoculation experiments. In each leaf segment, 100 infection sites were recorded.

Figure 4. Macroscopic phenotypes of interaction of SusBgt lines with Blumeria graminis f.sp. tritici ( Bgt ) and B. graminis f.sp. hordei-murini (Bghm ). (A) Development of colonies of Bgt on SusBgt SC and SusBgt DC at 8 dai. ( B)

Hypersensitive reaction of SusBgt SC to Bghm at 8 dai. Vada is immune to both ff.spp.

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The majority of those germlings that had formed a primary haustorium were stopped at the secondary penetration attempts. In both SusBgt SC and SusBgt DC , a considerable proportion of successfully penetrated germlings succeeded in establishment of a secondary haustorium (44 and 42%, respectively, Figure 5D). Interestingly, the secondary and subsequent haustoria were almost always detected in the same cell in which the primary haustorium had been established, typically resulting in two to five haustoria in one epidermal cell (Figure 6E). Subsequent penetration attempts in epidermal cells located away from the first infected cell usually failed (Figure 6F). At 8 dai, lines differed in the proportion of established germlings that had proceeded to the formation of at least some conidiophores. This conidiation was most conspicuous (34% of those germlings that had formed a haustorium) in

SusBgt SC , and much less so in SusBgt DC (6%) (Figure 5G). In the parental line Trisuli Bazar 9, a similar proportion of established germlings reached conidiation as in

SusBgt SC (Figure 5G), but the rate of establishment was lower than in SusBgt SC (Figure 5A). In summary, the results showed that SusBgt lines show an exceptional susceptibility to the non-adapted Bgt , and the amenability to formation of haustoria is not necessarily associated with amenability to conidiation. Both lines are valuable to study the inheritance of non-host resistance in barley to powdery mildews.

The level of susceptibility of SusBgt lines to other non-adapted powdery mildew pathogens To determine whether the SusBgt lines are susceptible to other non-adapted powdery mildew pathogens, the four parental lines, SusBgt lines and the reference barley cv. Vada were inoculated with one isolate each of other non-adapted formae speciales and species of powdery mildew pathogens.

SusBgt lines and their parental lines showed relatively high haustorium formation and conidiation by the powdery mildew fungus of Hordeum murinum, Bghm (Figure

5B, 5E and 7B). At the macroscopic level, SusBgt SC showed a remarkably strong HR reaction at 8 dai (Figure 4B). All tested accessions allowed at least some haustorium formation and conidiation by the rye powdery mildew pathogen ( Bgs) (Figure 5C and 5F), but the SusBgt lines were not more susceptible to this mildew form than the other lines were. SusBgt SC and SusPtrit that allowed relatively high establishment by Bgt (>50 %) allowed considerably less establishment by Bgs (< 26 %). Surprisingly, Vada, which did not allow any haustorium formation by Bgt , allowed relatively high haustorium formation by Bgs (31 %) . Relatively few germlings of Bgs that had formed a first haustorium proceeded to form a secondary haustorium (< 22%) and conidia (<14%) compared to Bgt (< 45 and < 35%, respectively) (compare Figure 5D with

70 New perspectives to identify genes that…

Figure 5F, and note the difference in the scale of the ordinates) . The tested barley lines were all nearly immune to the mildew fungi of oat, Bromus mollis and Dactylis glomerata. At most, 10% of the germlings formed a haustorium, and none of the germlings formed conidia. Both SusBgt lines allowed some haustorium formation (35- 51%) by the Agropyron powdery mildew ( Bgar ), but the rate of established germlings reaching conidiation was considerably higher in SusBgt SC than in SusBgt DC (80% and 21%, respectively) (data not shown). As with the Bgt , during infections by the other non-adapted mildew strains, except Bghm (Figure 7A), second and subsequent haustoria were almost always formed in the same cell as the first haustorium, and failed penetration attempts along the spreading hyphae were commonly observed. The two additional non-adapted mildew species, O. neolycopersici and Sphaerotheca fuliginea , were not observed to form any haustoria on any of the barley accessions.

Discussion Although we have not yet started to perform formal inheritance studies, several interesting conclusions on the genetic basis of the basal resistance of barley to non- adapted Bg mildews have emerged. 1-The resistance has a quantitative inheritance, as suggested by the quantitative differences between accessions and by the transgressive segregation when combining the parental lines. Our results suggest that crossing and selection among some barley accessions with some level of susceptibility resulted in the accumulation of susceptibility factors (or depletion of resistance factors) to the non-adapted wheat powdery mildew, Bgt . These lines showed an unprecedented level of haustorium formation and colony development by this non-adapted target mildew. We conclude that non-host resistance of barley to Bgt is based on several minor genes, each with a small effect. 2-Genes for basal resistance have pathogen developmental stage-specific effects. If quantitative trait loci (QTLs) indeed represent operative targets for pathogen-delivered effectors to silence aspects of the defense (Jafary et al ., 2006; Niks & Marcel, 2009), silencing of the defense at the first penetration stage may require different effector- target interactions than ESH development or conidiation. Also, it seems that making the first haustorium (= silencing the defense of the first plant cell) does not imply that the defenses of the next cells are silenced easily as well. Arabidopsis penetration mutants ( pen ) allow higher frequencies of penetration by the non-adapted powdery mildew pathogen (Bgh ), but other genes ( Sag101 , Pad4 ) compromise subsequent stages in the infection process (Lipka et al ., 2005). Consonni et al . (2006) reported

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A Bgt B Bghm C Bgs 100% 90% 100% 100% 80% 90% 90% SPP-HR 70% 80% 80% 60% 70% 70% 50% 60% 60% 50% 50% SPP+HR 40% 40% 40% 30% 30% 30%

Interaction sites Interaction sites 20% 20% 20% FPP-HR 10% sites Interaction 10% Interaction sites 10% 0% 0% 0% FPP+HR

9 2 t y 9 2 t it o r ri l r ri C r 9 r C g a SC DC ant o a t D a e 2 t SC D ada z Pt t t Vada iv pr z Vada z t V Ro s V i Hiproly hame Su H hame SusP Hiproly ham SusP C li Ba C li Ba C usBg SusBg SusBg u SusBgt SC SusBgt S SusBgt suli Ba s isu Tri Tri Tr

E D Bgt Bghm F Bgs

50 40 30 40 35 30 25 30 25 20 20 15 20 15 10 10 10 5 5 Interaction sites [%] sites Interaction Interaction sites [%] sites Interaction 0 0 Interaction [%] sites 0

2 2 a 9 rit C C ly 9 2 it C C a r e SC SC o r e tr S D d a t D roly D r a m P t t a p ame Vad ip az a s g g V Hiproly ham Hi h H B h u B B C SusPt sBgt C SusPtrit usBgt sBgt i C S s s li Baz S l u u SusBg Su suli Bazar 9 Su su S S isu i ri Tr Tr T

H G Bgt Bghm I Bgs

40 100 90 30 30 80 25 70 60 20 20 50 15 40 10 30 10 20 10 5 Interactionsites [%] Interactionsites [%] 0 0 Interactionsites [%] 0

2 y 9 C da ly 9 2 it C C a ol r D o r e tr S D d t SC DC r a m P t t a sPtrit g gt Vada pr Va ip az a s g g V Hiproly u sB B Hi H B h u B B Chame S s Chame 2 SusPtrit usBgt SC i C S s s S usBgt l u u Su Su uli Baza S su S S ris ri Trisuli Bazar 9 T T Figure 5 . Interaction of SusBgt lines and the parental lines with three non-adapted Bg forms. Three upper figures ( A-C): defense reactions at 72 hai. White-dotted columns (FPP+HR) represent failed primary penetration with HR. White columns (FPP-HR) represent failed primary penetration without HR. Black columns (SPP+HR) represent the successful primary penetration with a haustorium and with HR. Cross-hatched columns (SPP-HR) represent the successful primary penetration with a haustorium but without HR. Data represent the means of four leaf segments collected from two independent inoculation experiments. In each leaf segment, 100 infection sites were recorded. Vivant is a wheat cultivar, and Rogo is a rye cultivar (Each is susceptible to its own mildew forms). Three middle figures ( D-F): development of secondary haustoria in the SPP infection units of A-C. Black-dotted columns represent the percentage of infection units that developed at least one secondary haustorium at 72 hai. Three lower figures ( G-I): development of conidia. The gray columns represent the percentage of established infection units that formed at least one conidiophore at 8 dai. Data represent the means of four or two leaf segments collected from two independent inoculation experiments.

72 New perspectives to identify genes that…

Figure 6. Interaction of SusBgt lines with non-adapted Bgt mildew fungus. ( A) A germling that stopped at the penetration stage in an epidermal cell, which shows an H 2O2 reaction in the whole cell, indicating HR. ( B) A failed germling that stopped at the penetration stage in association with papilla formation. ( C) A successfully penetrating germling that elicited HR. ( D) A successfully established germling that formed elongated secondary hyphae (ESH). ( E) Multiple haustorium formation (arrows) in an epidermal cell where the primary haustorium was formed. ( F) Failed secondary penetrations (arrows) along an ESH. ( G) An established colony of Bgt at 8 dai on SusBgt DC that did not result in conidiation. ( H) Abundant conidiation by an established colony of Bgt at 8 dai on SusBgt SC . The scale bar represents 100 m.

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Figure 7. Haustorium formation and conidiation of non-adapted Bghm and Bgar mildews on SusBgt

lines at 8 dai. ( A) A successfully established germling of Bghm on SusBgt SC that resulted in formation of a hyphal network and several secondary haustoria (arrows). ( B) Abundant conidiation by an

established colony of Bghm at 8 dai on SusBgt SC . ( C) An established colony of Bgar at 8 dai on

SusBgt DC that did not result in conidiation. ( D) Abundant conidiation by an established colony of Bgar

at 8 dai on SusBgt SC . The scale bar represents 100 m. that the Arabidopsis Atmlo2 pen1 double mutant allows more cell entry by Golovinomyces cichoracearum , but no significant increase in conidiophore production. For those later stages of development, apparently EDS1 , SAG101 and PAD4 need to be silenced. 3-Genes for basal resistance are mildew forma specialis -specific. SusBgt lines and SusPtrit are remarkably accessible to Bgt and allow relatively high frequencies of fungal establishment. Yet, they show a relatively high level of basal resistance to the adapted powdery mildew pathogen, Bgh. Vada had a high level of resistance to Bgt , but was not equally resistant to the rye mildew Bgs . The factors in SusBgt DC that hampered conidiation by Bgt compared to SusBgt SC (Figure 5G) seem to also be effective against Bgar (Figure 7C and D), but not against Bghm (Figure 5H and 7B) or Bgs (Figure 5I). Selection for high susceptibility to Bgt has not affected the level of resistance to Bga and other non-adapted mildews. Our results in the present study show many parallels with the barley–rust interaction (Atienza et al ., 2004; Jafary et al ., 2006; Marcel et al., 2007; Jafary et al ., 2008). In

74 New perspectives to identify genes that…

both pathosystems, accumulation of genes for susceptibility to non-adapted pathogen forms and species appears feasible. Non-host resistance to rust species and to non- adapted formae speciales of powdery mildew seems similar to quantitative, basal host resistance, and the resistance mechanism in both pathosystems is mainly based on non-hypersensitive mechanisms. Powdery mildew pathogens should suppress PAMP-triggered immunity (PTI) to establish basic compatibility. This suppression of PTI requires compatibility between the effectors and the operative targets in the plant. Effectors (O’Connell & Panstruga, 2006) and their operative targets in the plant (van der Hoorn & Kamoun, 2008) both seem to be under strong diversifying selection. For each plant species, a different bouquet of effectors may be required to suppress PTI (Almeida et al ., 2009; Niks & Marcel, 2009), implying that each mildew form probably has a different set of effectors, each tuned to the operative targets in their host plant species. Depending on the fungal development stage, different aspects of defense should be reprogrammed, again requiring different effectors. By common evolutionary ancestry, however, some compatible variants of operative target motifs may occur in some non- host plant accessions. Non-adapted mildews cannot be expected to suppress PTI of barley effectively, but, in the SusBgt lines, we may have accumulated variants of the targets that can be suppressed relatively easily by Bgt . New evidence has emerged on the involvement of some candidate genes in the basal host and non-host resistance of barley against mildew pathogens (Douchkov et al ., 2005; Miklis et al ., 2007). An example of such a candidate gene is HvSNAP34, which plays a role in basal host and non-host resistance of barley to Bg . Transient- induced gene silencing (TIGS) of this candidate gene strongly increased the rate of successful haustorium formation of both adapted ( Bgh ) and non-adapted powdery mildew fungi ( Bgt ) (Douchkov et al ., 2005). HvSNAP34 could be one of the operative targets of effectors to enhance successful establishment of haustoria by invading mildew fungi. We are now developing mapping populations of SusBgt SC × Vada and

SusBgt DC × Vada to map the QTLs (genes) that determine the level of basal resistance to the various mildew forms used in this study, similar to what was done for rusts in Jafary et al. (2006). This will enable us to identify the targets involved in non- host immunity in barley against mildew pathogens. Finally, the existence of relatively high levels of susceptibility of SusBgt lines to more than one forma specialis of Bg, may facilitate the cross-hybridization between different ff. spp. of Bg , as done on the shared host Triticum urartu for crossing f.sp. tritici with f.sp. agropyri by Tosa (1989) . Such pathogen and plant inheritance studies can contribute considerably to our

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understanding of the evolutionary and genetic basis of the host-pathogen interactions in Gramineous powdery mildew pathosystems.

Acknowledgements Anton Vels is gratefully acknowledged for his excellent technical assistance in greenhouse experiments. We thank Dr. Patrick Schweizer (Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Germany) for providing the wheat isolate of mildew. Bert Essenstam from the Unifarm of Wageningen University is acknowledged for providing the cucumber powdery mildew and multiplication of wheat powdery mildew. We thank Dr. Harold E. Bockelman (National Small Grains Collection, USDA, USA) for providing the seeds of barley cv. Turkey 290, the Centre for Genetic Resources, the Netherlands (CGN) for proving the seeds of 54 accessions of barley, and Dr. Rajeev K. Varshney from IPK, Germany, for providing the seeds of 227 accessions of barley (ICARDA collection) used in this study. ICARDA (Syria) is acknowledged for its indirect contribution by preparing the seeds. We thank Dr. Viktor Korzun and Clemens Springmann (PLANTA Angewandte Pflanzengenetik und Biotechnologie, Germany) for making the DHs. We thank Prof. Richard G.F. Visser for critically reading the manuscript and for his continuous support.

References Atienza S, Jafary H, Niks RE. 2004. Accumulation of genes for susceptibility to rust fungi for which barley is nearly a nonhost results in two barley lines with extreme multiple susceptibility. Planta 220: 71-79. Block A, Li G, Fu Z, Alfano JR. 2008. Phytopathogen type III effector weaponry and their plant targets. Current Opinion in Plant Biology 11: 396-403. Carver TLW, Lyngkjær MF, Neyron L, Strudwicke CC. 1999. Induction of cellular accessibility and inaccessibility and suppression and potentiation of cell death in oat attacked by Blumeria graminis f.sp. avenae . Physiological and Molecular Plant Pathology 55: 183–196. Chisholm ST, Coaker G, Day B, Staskawicz BJ. 2006. Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124: 803-814. Collinge DB, Jensen MK, Lyngkjaer MF, Rung J. 2008. How can we exploit functional genomics approaches for understanding the nature of plant defences? Barley as a case study. European Journal of Plant Pathology 121: 257-266. Consonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, Westphal L, Vogel J, Lipka V, Kemmerling B, Schulze-Lefert P, et al. 2006. Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nature Genetic 38: 716–720. Douchkov D, Nowara D, Zierold U, Schweizer P. 2005. A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. Molecular Plant- Microbe Interactions 18: 755-761. Eichmann R, Schultheiss H, Kogel KH, Hückelhoven R. 2004. The barley apoptosis suppressor homologue bax inhibitor-1 compromises nonhost penetration resistance of barley to the inappropriate pathogen Blumeria graminis f.sp tritici. Molecular Plant-Microbe Interactions 17: 484–490. Eshed N, Wahl I. 1970. Host ranges and interrelations of Erysiphe graminis hordei , E. graminis tritici , and E. graminis avenae . Phytopathology 60: 628-634. Hardison JR. 1944. Specialization of pathogenicity in Erysiphe graminis on wild and cultivated grasses. Phytopathology 34: 1–20. Heath MC. 2000. Nonhost resistance and nonspecific plant defences. Current Opinion in Plant Biology 3: 315-319. Hückelhoven R. 2007. Cell wall-associated mechanisms of disease resistance and susceptibility. Annual Review of Phytopathology 45: 101–127. Hückelhoven R, Dechert C, Kogel KH. 2001. Non-host resistance of barley is associated with hydrogen peroxide burst at sites of attempted penetration by wheat powdery mildew fungus. Molecular Plant Pathology 2: 199-205.

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Jafary H, Albertazzi G, Marcel TC, Niks RE. 2008. High diversity of genes for nonhost resistance of barley to heterologous rust fungi. Genetics 178: 2327–2339. Jafary H, Szabo LJ, Niks RE. 2006. Innate nonhost immunity in barley to different heterologous rust fungi is controlled by sets of resistance genes with different and overlapping specificities. Molecular Plant–Microbe Interactions 19: 1270–1279. Koga H, Bushnell WR, Zeyen RJ. 1990. Specificity of cell type and timing of events associated with papilla formation and the hypersensitive reaction in leaves of Hordeum vulgare attacked by Erysiphe graminis f.sp. hordei . Canadian Journal of Botany. 68: 2344–2352. Lin MR, Edwards HH. 1974. Primary penetration process in powdery mildewed barley related to host cell age, cell type, and occurrence of basic staining material. New Phytologist 73: 131-137. Lipka V, Dittgen J, Bednarek P, Bhat R, Wiermer M, Stein M, Landtag J, Brandt W, Rosahl S, Scheel D, et al. 2005. Pre- and postinvasion defences both contribute to nonhost resistance in Arabidopsis. Science 310: 1180-1183. Mains EB, Dietz SM. 1930. Physiologic forms of barley mildew, Erysiphe graminis hordei Marchal. Phytopatlogy 20: 229-239. Marcel TC, Varshney RK, Barbieri M, Jafary H, de Kock MJD, Graner A, Niks RE. 2007. A high- density consensus map of barley to compare the distribution of QTLs for partial resistance to Puccinia hordei and of defence gene homologues. Theoretical and Applied Genetics 114: 487– 500. Menzies JG, MacNeill BH. 1989. Infection of species of the Gramineae by Erysiphe graminis f.sp. hordei and Erysiphe graminis f.sp. tritici. Canadian Plant Disease Survey 69: 105-108. Miklis M, Consonni C, Bhat RA, Lipka V, Schulze-Lefert P, Panstruga R. 2007. Barley mlo modulates actin-dependent and actin-independent antifungal defense pathways at the cell periphery. Plant Physiology 144: 1132–1143. Mysore KS, Ryu CM. 2004. Nonhost resistance: how much do we know? Trends in Plant Science 9: 97-104. Niks RE, Marcel TC. 2009. Non-host and basal resistance: how to explain specificity? New Phytologist 182: 817–828. Nurnberger T, Brunner F, Kemmerling B. Piater L. 2004. Innate immunity in plants and animals: striking similarities and obvious differences. Immunological Reviews 198: 249-266. O'Connell RJ, Panstruga R. 2006. Tête à tête inside the plant cell: establishing compatibility between plants and biotrophic fungi and oomycetes. New Phytolologist 171: 699-718. Olesen KL, Carver TLW, Lyngkjær MF. 2003. Fungal suppression of resistance against inappropriate Blumeria graminis formae speciales in barley, oat and wheat. Physiological and Molecular Plant Pathology 62: 37-50. Peterhänsel C, Freialdenhoven A, Kurth J, Kolsch R, Schulze-Lefert P. 1997. Interaction analyses of genes required for resistance responses to powdery mildew in barley reveal distinct pathways leading to leaf cell death. Plant Cell 9: 1397–1409. Schweizer P. 2007. Nonhost resistance of plants to powdery mildew-New opportunities to unravel the mystery. Physiological and Molecular Plant Pathology 70: 3–7. Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB. 1997. Subcellular localization of H 2O2 in plants: H 2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant Journal 11: 1187–1194. Thordal-Christensen H. 2003. Fresh insights into processes of non-host resistance. Current Opinion in Plant Biology 6: 351-357. Tosa Y, Shishiyama J. 1984. Defense reactions of barley cultivars to an inappropriate forma specialis of the powdery mildew fungus of gramineous plants. Canadian Journal of Botany 62: 2114–2117. Tosa Y. 1989. Genetic analysis of the avirulence of wheatgrass powdery mildew fungus on commons wheat. Genome 32: 913-917. Trujillo M, Troeger M, Niks RE, Kogel KH, Hückelhoven R. 2004 . Mechanistic and genetic overlap of barley host and non-host resistance to Blumeria graminis . Molecular Plant Pathology 5: 389- 396. van der Hoorn RAL, Kamoun S. 2008. From guard to decoy: a new model for perception of plant pathogen effectors. Plant Cell 20: 2009-2017. Wolf G, Fric F. 1981. A rapid method for staining Erysiphe graminis f.sp. hordei in and on whole barley leaves with a protein-specific dye. Phytopathology 71: 596-598.

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73 Rustinduced powdery mildew resistance

Rust-induced powdery mildew resistance in barley

R. Aghnoum, R.G.F. Visser, P. Schweizer and R.E. Niks

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Abstract Different pathogens employ different strategies to suppress their host defense mechanisms. We observed that a compatible interaction of barley-Puccinia hordei induced increased papilla-based resistance to a challenge infection by a compatible powdery mildew isolate despite the different strategies of suppression of defense by the two organisms. The level of rust-induced mildew resistance varies among barley accessions and is not determined by the virulent/avirulent spectra of the challenger isolate. Our histological data suggest that the inducer effect is due to rust-plant communication during stoma penetration by the rust fungus, or to the presence of the rust hyphae in the apoplast, or to some degree of pre-haustorial resistance mounted by the mesophyll cells to the invading rust fungus. Macroarray gene expression analysis showed that several genes related to metabolism and photosynthesis were highly down regulated and a few genes involved in plant defense including a pathogenesis related protein (PR-1), and Cysteine synthase were highly up regulated in the double inoculated treatment. Both our histological and gene expression analysis suggest that the rust “primes” the basal mildew resistance genes prior to the challenge mildew infection.

Introduction Infection by a pathogen or parasite may condition plants to activate or suppress defense reactions against subsequent infection attempts by other pathogen strains or species. Induction of plant defense systems as a result of prior exposure to pathogens (Ryals et al. , 1996), non-pathogenic root-colonizing bacteria (van Loon et al ., 1998), feeding by herbivores (Kessler & Baldwin, 2002) or treatment with chemicals (Oostendorp et al ., 2001) is termed induced resistance. Three types of induced resistance have been described, viz. systemic acquired resistance (SAR) (see Glossary) (Durrant & Dong, 2004), induced systemic resistance (ISR) (see Glossary) (van Loon et al ., 1998) and induced localized resistance (Kunoh, 2002). Suppression of plant defense systems against a non-adapted or incompatible race of a pathogen as a result of prior exposure to a compatible pathogen is called induced susceptibility. Induced localized resistance and induced susceptibility have been reported in many plant-pathogen combinations (Table 1). In cereals induction of localized susceptibility or resistance has been reported as a result of a prior attack by both necrotrophic and biotrophic fungi (e.g. Jørgensen et al., 1998; Kunoh et al ., 1988; Lyngkjær & Carver, 1999; Niks, 1989). In cereals, most research on localized induction of resistance and susceptibility has concentrated on interaction of compatible and incompatible isolates

80 Rustinduced powdery mildew resistance

of the powdery mildew pathogen, Blumeria graminis (hereafter Bg ). Failed establishment (see Glossary) by an adapted or non-adapted powdery mildew pathogen induces penetration resistance to subsequent challenge inoculation with a compatible isolate (e.g. Hwang & Heitefuss, 1982; Cho & Smedegaard-Petersen, 1986; Carver et al ., 1999; Lyngkjær & Carver, 1999; Kunoh, 2002). Conversely, successful penetration and haustorium formation by an adapted, virulent isolate of Bg induces susceptibility at cell level to haustorium formation by subsequent challenge with adapted virulent or avirulent isolates and also non-adapted formae speciales and species (e.g. Tsuchiya & Hirata, 1973; Carver et al ., 1999; Lyngkjær et al ., 2001; Olesen et al ., 2003). “Susceptibility” at cell level is often called accessibility, because the avirulent or non-adapted challenger pathogen typically will not complete its life cycle, due to defense mechanisms that back-up the penetration resistance. For similar reasons “resistance” at cell level is often called inaccessibility. Most of the reports indicate that this induction of accessibillity and inaccessibility is localized and limited to the challenged cell and its adjacent cells. Induced resistance to rust pathogens as a result of prior inoculation with the same or different rust pathogens has been reported by several workers (e.g. Kochman & Brown, 1975; Sebesta et al ., 1996; Calonnec, et al . 1996; Pei et al . 2003). In the barley leaf rust ( Puccinia hordei ) system, failed haustorium formation by an non- adapted rust fungus, however, does not induce reduced haustorium formation by a compatible challenger rust (Niks, 1989) but a compatible interaction suppresses the defense system of the same cell at challenge with a non-adapted rust species. Powdery mildew and rust fungi both have biotrophic lifestyles and require living plant tissues to survive and reproduce. Successful establishment of specialized feeding structures of the pathogen, the so-called haustoria, is a prerequisite for a compatible interaction. In barley, non-host resistance against non-adapted powdery mildews, the quantitatively inherited basal resistance and mlo -mediated resistance to the adapted powdery mildew all act on the basis of papilla formation at the point of attempted ingress into the cell, which results in impaired haustorium formation. Papilla formation is involved in induced inaccessibility in the barley-powdery mildew (Sahashi & Shishiyama, 1986). A similar papilla formation is involved in basal and non-host resistance against rust fungi (Niks, 1986, 1989). Mellersh & Heath (2001) reported evidence that mildews and rust fungi follow different strategies to successfully suppress plant defense, and hence induce accessibility of plant cells. Rust fungi should decrease adhesion between the cell wall and the plasma membrane at the cell wall penetration point, but for successful infection by mildew no such decreased adhesion is required. We set out to determine

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whether a compatible rust interaction would induce increased accessibility to mildew, despite the different strategies of suppression of defense by the two organisms. We found, however, in exploring experiments, that compatible infections by Puccinia hordei induced increased papilla based resistance rather than decreased papilla based resistance to a challenge infection by a compatible powdery mildew isolate. In this paper we characterize this rust-induced mildew resistance at the cellular level. We also identified differentially expressed genes that can explain the strong papilla- based penetration resistance in double inoculated barley plants.

Materials and Methods Plant material Three barley near isogonics lines (Pallas, Pallas-Mla3 , Pallas-MlLa ), the mildew susceptible barley cv. ‘Manchuria’, two barley lines that possess high levels of basal resistance to leaf rust (Vada and 17-5-16), and three near-isogenic barley lines with different genotypes of MlLa and Rphq2 , the latter being a QTL for basal resistance to leaf rust (Marcel et al ., 2007) (Vada rphq2 mlLa , Vada rphq2 MlLa , and Vada Rphq2 mlLa ), were used to study the effect of pre-inoculation with leaf rust on powdery mildew development. Three seeds of each genotype were sown in trays of 37×39 cm in two rows on the right and left side of each tray. Plants were grown in a greenhouse compartment where the temperature was set at 18-20°C with 16 hours of light and a relative humidity of about 70%.

Pathogen material The isolate 1.2.1 of Puccinia hordei was used as the ‘inducer’. This isolate is able to establish a compatible interaction with all tested barley lines. Two isolates of Blumeria graminis f.sp. hordei, isolate C15 and Wag.04, were used as ‘challenger’. Isolate C15 is avirulent to Pallas-MlLa but virulent to Pallas-Mla3. Wag.04 isolate is the reverse, virulent to Pallas-MlLa but avirulent to Pallas-Mla3 . Leaf rust isolate 1.2.1 was maintained on line L94, and the powdery mildew isolates on Manchuria. An isolate of Puccinia recondita , the rye leaf rust pathogen, freshly propagated on a susceptible rye genotype and an isolate of Puccinia antirrhini, the snapdragon rust, collected from naturally infected common snapdragon ( Antirrhinum majus ) in Wageningen were used in this study.

Inducer inoculation Ten days after sowing, the fully expanded first seedling-leaves were positioned horizontally, adaxial side up. In the preliminary experiments, prior to the inoculation

82 Rustinduced powdery mildew resistance

with rust, the middle part of each leaf was covered with a 3 cm strip of aluminum foil to keep that middle section free of rust infection, in order to determine whether the effect of inducer inoculation on the challenger is localized or systemic. In each tray only the plants of one row were inoculated by the inducer and the plants of the other row were covered to keep them free of rust. For each box, 6 mg of fresh urediospores of P. hordei isolate 1.2.1 were diluted ten times with Lycopodium powder and dusted over the plants in a settling tower. This inoculum dose results in about 350 urediospores per cm 2. Inoculated plants were incubated overnight in a dark chamber with 100% humidity and then transferred to a greenhouse compartment with the above mentioned conditions.

Challenger inoculation Five days after the inoculation with the inducer, plants were subjected to inoculation by the challenger mildew. A settling tower was used for inoculation. Freshly produced conidia of powdery mildew were blown into the tower over the horizontally fixed adaxial sides of the first leaves. The density of inoculum was adjusted to 100-150 conidia per cm 2. The inoculated plants were incubated in a growth chamber at 18-20 oC with fluorescent light irradiance (100 micromol/m2/s) supplied for 16 h per day.

Histological assessment of challenger infection Forty eight hour after inoculation (hai) of the challenger, a leaf segment of about 3 cm long was collected from the basal side of the rust-mildew inoculated portion and the whole rust-free middle portion. From the control plants a leaf segment of about 3 cm long was collected from the middle part of the leaves. From each genotype, two seedlings were sampled and one seedling was kept to monitor the efficiency of inoculation and infection type of the powdery mildew at macroscopic level. Infection types (IT) of powdery mildew were scored based on the 0–4 scale proposed by Mains & Dietz (1930) on those plants that were only inoculated with the challenger. The leaf segments were submerged in a solution of acetic acid-ethanol (3/1 v/v) at least for 3 hours to be cleared. They were stained by boiling for 8 min in a solution of alcoholic lactophenol (96% ethanol-lactophenol 1/1 v/v) containing 0.1 mg ml-1 Trypan Blue and then cleared in a Chloral Hydrate solution (2.5 mg ml-1) overnight (Peterhänsel et al. , 1997). Those infection units that were stopped at penetration stage in association with papilla deposition were considered failed primary penetration (FPP) and infection units that succeeded to penetrate the epidermal cells and established a haustorium and an elongated secondary hypha (ESH) were

83 New perspectives to identify genes that…

Table 1: A summary of published literature on pathogen-induced susceptibility and resistance Plant-inducer Plant-challenger Outcome of Crop Inducer Challenger 1 Reference(s) interaction interaction interaction Bean Uromyces phaseoli Uromyces phaseoli S S IR Yarwood 1954 Melon Sphaerotheca fuliginea Bgh 4, Bgt 4 S nhR 3 IS Oku & Ouchi 1976 Pyricularia oryzae Pyricularia oryzae R S IR Rice Manandhar et al ., 1998 Bipolaris sorokiniana Pyricularia oryzae nhR S IR Puccinia triticina P. coronata avenae nhR S IR Oat Kochman & Brown 1975 P. graminis tritici P. graminis avenae nhR S IR S Oat Bga 4 Bga S IS S Carver et al ., 1999; Lyngkjær & Carver 2000 Bga Bga R IR

nhR Oat Bga Bgh,Bgt S IS Olesen et al ., 2003

IR Wheat Puccinia striiformis P. striiformis R S Calonnec et al., 1996

Wheat Septoria tritici Septoria tritici R S IR Halperin et al ., 1996 nhR Bgt Bgh,Bga S IS Olesen et al ., 2003 Wheat S Bgh Bgt R IR Schweizer et al ., 1989

Barley F. oxysporum f.sp. radicis-lycopersici Bgh nhR S ISR 2 Nelson 2005 Barley Cladosporium macrocarpum Bgh Saprophyte S IR Gregersen & Smedegaard, 1989 nhR Puccinia hordei Prr S IS Niks, 1989 Barley S P. recondita f.sp. recondita(Prr) P. hordei nhR No IR

Bipolaris maydis Drechslera teres nhR S IR Barley Jørgensen et al., 1998 Septoria nodorum Drechslera teres nhR S IR Barley Bgh Sph. fuliginea S nhR IS Oku & Ouchi, 1976 Barley Bgh Erysiphe pisi S nhR IS Kunoh et al ., 1988; Kunoh, 2002 Barley Bgh Various PM fungi S nhR IS Tsuchiya & Hirata, 1973 Barley Bgh Bgt,Bga S nhR IS Ouchi et al ., 1976a; Olesen et al ., 2003 Barley Erysiphe pisi Bgh nhR S IR Kunoh et al ., 1988; Kunoh, 2002 Ouchi et al ., 1976a; Ouchi et al ., 1976b; Gregersen and Barley Bgt Bgh nhR S IR Smedegaard 1989 Bgh Cho and Smedegaard-Petersen, 1986; Lyngkjær & Barley Bgh R S IR Carver, 2000; Lyngkjær et al ., 2001 Barley Bgh Chin et al ., 1984; Lyngkjær et al ., 2001; Lyngkjær & Bgh S S /R IS Carver, 2000 1 Induced resistance (IR) and induced susceptibility (IS) have been used for the effect of inducer on challenger development on entire leaves, whole plants or at cell level 2 ISR, Induced systemic resistance; 3nhR, non-host resistance; 4Bga , Bgh and Bgt stand for Blumeria graminis f.sp. avenea , hordei and tritici respectively

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Rustinduced powdery mildew resistance considered as successful primary penetration (SPP) (Asher & Thomas, 1983). Because different types of epidermal cells show different reactions to powdery mildew infection (Lin & Edwards, 1974) only stomatal and interstomatal cells (type A and type B epidermal cells following the terminology of Koga et al., 1990) were examined, and long epidermal cells over-lying vascular tissues (type C) were not considered . In each leaf segment 150 powdery mildew germlings were scored. Only those epidermal cells attacked by a single mildew fungal germling were considered. Preparations were examined at a magnification of 400 × using a Zeiss Axiophot microscope, using transmitted white light .

Transcriptome analysis of rust-induced mildew resistance In order to study defense-related gene expression in barley that was double inoculated with rust and powdery mildew, a macroarray expression analysis was carried out. Barley line 17-5-16 (Parlevliet & Kuiper, 1985) which showed in our histological experiments a high level of rust-induced penetration resistance to powdery mildew was selected for this gene expression study. Treatments were as follows: treatment 1, inducer ( P. hordei , isolate 1.2.1.) + challenger ( Bgh , isolate C15); treatment 2, only inducer; treatment 3, only challenger; treatment 4, mock, no inoculation, only Lycopodium powder applied as for the other treatments. About 60 plants were grown in each box (37 × 39 cm). For each type of treatment one box was used. Ten days after sowing, first formed leaves were fixed in a horizontal position, with the abaxial side up. The inoculation procedure, the density of inducer and challenger and the incubation conditions were as in the previous experiments. Twelve hai, 15 leaves were sampled per treatment. Leaf samples were shock-frozen in liquid nitrogen immediately and stored at –80 o C. The experiment was carried out in three independent inoculation experiments in a randomized block design .

The barley PGRC2 cDNA array We used the barley PGRC2 cDNA macroarray, which is an extended version of the PGRC1 array described by Schweizer (2008). These cDNA arrays have been developed at IPK (Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany) and contain approximately 13,000 and 10,000 unigenes respectively, spotted on two membranes.

RNA isolation Total RNA was extracted from whole-leaf material sampled at 12 hai. About 10 leaf samples were ground in liquid nitrogen and about 100 mg of the homogenized tissue

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samples was used for RNA extraction with TRIZOL ® Reagent according to the manufacturer’s protocol with some modification of the procedure, viz. the aqueous phase was extracted twice with chloroform.

Synthesis of 33 P-labelled cDNA and hybridization procedures mRNA was extracted from 37.5 g of the total RNA using Dynal magnetic beads according to the manufacturer’s protocol. 33 P-labelled second-strand cDNA probes were prepared as described by Sreenivasulu et al . (2002). cDNA arrays were first pre-hybridized and then hybridized with cDNA probes as described by Sreenivasulu et al . (2002). The hybridization period was prolonged to 18 h. as suggested by Zierold et al . (2005).

Spot detection and data normalization The hybridized membranes were scanned by the phosphorimager (Fuji BAS3000 reader). Spots were detected and spot intensities were quantified by using the ArrayVision 8.0 software package (Imaging Research Inc., St. Catharines, Ontario, Canada). Signal intensities were normalized after background subtraction as described by Sreenivasulu et al. (2002). Only unigenes with a signal/background ratio of more than 2.0-fold in at least two hybridizations were considered. Differentially regulated genes were identified by calculating the Q value (false discovery rate) by using the EDGE (Extraction of Differential Gene Expression) software (Leek et al . 2006). Transcripts that showed a Q-value of less than 0.2 (FDR<20%) and a p-value of less than 0.05 between sample pairs were selected for further analysis.

Results Effect of prior infection with leaf rust on powdery mildew development Induction of localized inaccessibility to powdery mildew by inducer The leaf rust fungus (inducer) was able to establish a compatible interaction with all tested barley genotypes. At five days after inoculation (dai) with the inducer, when the plants were subjected to the attack by challenger, the leaf rust had formed pale green flecks and at 8 dai mature rust pustules had formed. On the control plants that were only inoculated with challenger mildew, depending on the barley genotype, 23 to 41% of the infection units were stopped at penetration stage in association with papilla formation. When the challenger isolate was avirulent to the accession that carry corresponding R gene, the successfully penetrated host cells died in response to the attack. The inducer inoculation caused a significant

Rustinduced powdery mildew resistance reduction in successful haustorium formation by the challenger mildew in all tested barley accessions, but the level of induced resistance differed between the accessions (Figure 2). The strongest effect of induced resistance was observed in the interaction of Pallas-Mla3 with the Wag.04 mildew isolate. On Pallas-Mla3 plants pre-conditioned by the inducer, only 9% of germlings succeeded to penetrate and form a haustorium, versus 58 % on the control plants. In the very mildew susceptible accession Manchuria, the lowest effect of induced resistance was observed. Data obtained from the middle parts of the leaves that were covered during inducer inoculation, indicated that this induction of penetration resistance hardly extends to tissue away from the rust infections, and therefore is hardly or not systemic (Figures 1 and 2A).

Figure 1. Rust-induced mildew resistance phenotype on barley line Pallas inoculated with leaf rust and powdery mildew (wag.04 isolate). Colonies of powdery mildew are visible on the right part of the leaf that only was inoculated with powdery mildew; on the left side, that was inoculated with leaf rust 5 days before mildew inoculation, hardly any colony is visible.

Induced inaccessibly is not determined by the virulent/avirulent spectra of the challenger isolate Because of the different levels of induced resistance on the three Pallas near- isogenic lines (Figure 2A), an obvious question to be answered was whether the virulence/avirulence of the challenger isolate (powdery mildew) to the Pallas near- isogenic lines that carrying the corresponding R gene, can influence the level of induced resistance. Similar results were obtained when we used another powdery mildew isolate (C15) that reacted differentially with the two near-isogenic lines (Pallas-Mla3 and Pallas-MlLa ) in comparison with Wag.04 isolate (Figure 2B). This indicates that induced resistance is not determined by the virulent/avirulent spectra of the challenger isolate. We tested Vada recombinants with different genotypes regarding Rphq2 and MlLa. Rphq2 is an allele for higher basal resistance to P. hordei , rphq2 for lower basal resistance to this rust fungus. MlLa is an allele for race-specific incomplete

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hypersensitive resistance to powdery mildew, mlLa is the susceptibility allele. The Rphq2 and MlLa loci are linked, and recombinants between these loci have been developed in Vada background (Marcel et al ., 2007). The four combinantions of alleles on these two loci showed similar and rather low levels of induced resistance (Figure 3). This is preliminary evidence that the level of basal resistance to P. hordei does not determine the level of induced resistance to powdery mildew, and that mildew R-gene MlLa is not a determining factor either. We also tested barley experimental line 17-5-16, a barley line that possesses a very high level of basal resistance to leaf rust (Parlevliet & Kuiper, 1985). In this line the inducer caused a high level of penetration resistance to the mildew challenger (Figure 3).

A 100 90 80 a a 70 a a a a b 60 a a 50 40 b 30 b 20

Penetration efficiency [%] b 10 0 Pallas Pallas-Mla3 Pallas-MlLa Manchuria

B 100 90 80 70 a 60 a a a 50 a 40 b 30 b b 20 Penetration efficiency [%] 10 0 Pallas Pallas-Mla3 Pallas-MlLa Manchuria

Figure 2. Penetration efficiency of powdery mildew germlings on different barley lines at 48 hai. A, Wag.04 isolate, avirulent to Pallas-Mla3 but virulent to Pallas-MlLa . B, C15 isolate, avirulent to Pallas-MlLa but virulent to Pallas-Mla3. Gray-dotted columns represent the penetration efficiency of powdery mildew when barley leaves were inoculated with the inducer ( Puccinia hordei isolate 1.2.1) five days before inoculation with the challenger. White columns represent penetration efficiency in a 3 cm strip of leaves that was covered during inoculation with the inducer and were inoculated only with the challenger. Gray columns represent penetration efficiency on control plants that were inoculated only by powdery mildew at the same time as inoculation of other treatments with the challenger. In Figure 2A data of each column are based on an average of 600 mildew infection units collected from 4 leaf segments (two leaves in two experiments per accession/treatment combination). In Figure 2B data of each column are based on an average of 300 mildew infection units collected from 2 leaf segments each from an independent inoculation experiment. Means followed by the same letter within a genotype are not significantly different (p= 0.05).

Rustinduced powdery mildew resistance

Stoma penetration and intracellular hyphae may contribute to induced resistance We determined which aspect of rust infection may contribute to induced resistance. We applied a non-adapted rust, viz. the snapdragon rust fungus P. antirrhini . We checked the barley leaves inoculated with this rust at microscopic level. This rust produces only germ tubes on the barley leaf surface, and hardly or not penetrates through stomata. Although the snapdragon rust was applied at about two times higher inoculum dose than the leaf rust, we observed that it did not induce resistance against powdery mildew (Figure 4). This indicates that induced resistance is not due to communication between the rust germ tube and the barley epidermal cells. Next we applied the non-adapted rust P. recondita as inducer. P. recondita is able to find the stomata and penetrate through them, but hardly forms any haustorium in barley (Niks, 1989). We found that application of P. recondita reduced the haustorium formation by the mildew fungus from 47% in the control to 25% (Figure 4). There is a tendency that haustorium formation is not required for the induction of resistance to the mildew fungus, more replications are required to test whether this difference is significant. Application of the inducer inoculum P. hordei and challenger mildew on opposite leaf sides resulted in a similar level of induced penetration resistance to subsequent attack by powdery mildew as when both inoculations were applied to the adaxial leaf surface (Figure 4). This induction was not observed when P. recondita was applied on the opposite side of the leaf. This suggests that the rust intracellular hyphae and stoma penetration both may contribute to induced resistance.

Transcript profiling Out of approximately 13,000 unigenes spotted on the array, 6,053 unigenes showed hybridization signals at least 2.0 fold above the local background and they were selected for further statistical analysis. Only those unigenes that showed a Q-value less than 0.2 between sample pairs and P value < 0.05 were considered differentially regulated genes. In total, 285 gene/treatment combinations showed differential expression compared with the mock inoculation (Figure 5). A considerable set (67%) of differentially regulated genes in LR+PM was also differentially regulated in LR. Higher numbers of genes were found to be differentially regulated in LR+PM treatment (128) than in single inoculation treatments. On the other hand, direct comparison of gene expression between LR (Leaf Rust) and LR+PM (Leaf Rust + Powdery mildew) inoculations did not reveal any transcripts that were significantly

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[%] efficiency Penetration 10

0 (Rphq2 MlLa) (rphq2 mlLa) (rphq2 MlLa) (Rphq2 mlLa) 17-5-16

Basal resistance high low low high very high to P. hordei

Incomplete HR yes no yes no no to Bgh

Figure 3. Penetration efficiency of powdery mildew germlings (C15 isolate) on different near isogenic barley lines on the background of Vada and a line with high level of basal resistant to leaf rust (17-5-16). Gray- dotted columns represent the penetration efficiency of powdery mildew when barley leaves were inoculated with the inducer ( Puccinia hordei isolate 1.2.1) five days before inoculation with the challenger. Gray columns represent penetration efficiency on control plants that were inoculated only by powdery mildew at the same time as inoculation of other treatments with the challenger. Data of each column are based on an average of 300 infection units collected from 2 leaf segments (one leaf in two independent inoculation experiments per treatment). C15 isolate is avirulent on genotypes carrying the MlLa allele.

50 *

20 *

Penetration efficiency [%] efficiency Penetration 10

0 Only mildew P. hordei P. hordei P. recondita P. recondita Snapdragon downside downside rust

Figure 4. Penetration efficiency of powdery mildew germlings (Wag.04 isolate) on barley cv. Pallas inoculated with different inducer rusts. The inducers were inoculated five days before inoculation with the challenger. Data of each column are based on an average of 600 infection units collected from 4 leaf segments (two leaves in two experiments per treatment). * The mean difference is significant at 0.05 level (one sided test).

Rustinduced powdery mildew resistance different in their abundance, indicating the existence of quantitative rather than qualitative differences between induced but not challenged and induced-challenged leaves. Such differential expression patterns may have escaped detection due to the limited number of samples used for the analysis. To assess the quantitative difference in normalized signal intensities (Int) of differentially regulated genes between double inoculation treatment (LR+PM) and single mildew inoculation (PM) in a more robust manner, a differential index (DI; Zierold et al. , 2005) was calculated for each gene as follows:

DI = (Int LR+PM − Int PM )/ (Int LR+PM + Int PM ) DI > 0 indicates genes that on average show higher transcript abundance in LR+PM compared to PM. DI < 0 indicates genes that on average show higher transcript abundance in PM than in LR+PM. The genes with the greatest difference in expression level between LR+PM and PM showing a DI > 0.33 or DI < -0.33, which corresponds to a two-fold signal difference on average, are listed in Table 2. The results indicate that there are great differences in transcript abundance among the genes with different functions. The majority of the differentially regulated genes found to be involved in metabolism and photosynthesis and a few genes in disease resistance (Table 2 and Figure 6). Up-regulation of a pathogenesis related protein (PR-1), a marker for Systemic Acquired Resistance (SAR) and Cysteine synthase which is involved in balancing the production of ROS (reactive oxygen species) was observed in double inoculated treatment and in single rust treatment (Table 2, Figure 6).

Leaf rust (109) Powdery mildew (48)

23 1 14 30 55 3

Leaf rust + powdery mildew (128)

Figure 5. Venn diagram representation of overlapping and non-overlapping sets of differentially regulated genes (versus mock) between different treatments in barley line 17-5-16.

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Table 2. Differentially regulated genes in double inoculated ( leaf rust + powdery mildew) barley plants versus powdery mildew inoculated plants Clone ID Blast X description 1 Regulation DI 2

HD01N05 Pathogenesis-related protein up 0.97 HO16H10 PR-1, a pathogenesis related protein up 0.97 HO06C23 Protease inhibitor up 0.93 HW03J02 Hypothetical protein up 0.66 HO08E02 Cysteine synthase up 0.66 HO05I23 Ribulose-1,5-bisphosphate carboxylase down -0.33 HO03F06 (TPR)-containing protein down -0.34 HO05F14 Ribulose-1,5-bisphosphate carboxylase down -0.34 HB27P08 Phosphoethanolamine methyltransferase down -0.34 HO28H23 Fructose-bisphosphate aldolase down -0.34 HE01K15 Hypothetical protein down -0.36 GCW002K24 Unknown down -0.37 HO19N07 Chlorophyll a/b binding protein down -0.37 HO04E03 Ribulose-1,5-bisphosphate carboxylase down -0.38 HO02G01 Ribulose-1,5-bisphosphate carboxylase down -0.40 HZ48I22 Photosystem I antenna protein down -0.41 HM11H23 Photosystem I antenna protein down -0.42 HO01J24 Ribulose-1,5-bisphosphate carboxylase down -0.45 HB21N18 Chlorophyll a/b binding protein down -0.48 HO01D09 Chlorophyll a-b binding protein down -0.49 HE01E12 Chlorophyll a/b binding protein 2 down -0.50 HZ53A20 Metallothioneine type2 down -0.50 HO36J03 Carbonic anhydrase down -0.51 HA06N06 Fructose-bisphosphate aldolase down -0.51 HO38I13 Carbonic anhydrase down -0.52 HO02J18 Putative GTP-binding protein down -0.53 HG01D09 Carbonic anhydrase down -0.53 HA12L17 Phosphoglycerate kinase down -0.56 HV04H14 Putative thiamine biosynthesis protein down -0.57 HO28I13 Carbonic anhydrase down -0.57 HDP10O13 Photosystem II 10 kDa polypeptide down -0.76 HG01P09 Unknown down -0.80 HV03C20 Metallothionein-like protein type 3 down -0.80 HD08N06 Metallothionein-like protein down -0.81 1Description of the most significant Blast X hit (the lowest E value). 2Differential index of transcript abundance between double inoculated and single mildew inoculated plants. Only genes that showed at least 2-fold difference in transcript abundance were presented.

Rustinduced powdery mildew resistance

Discussion Our histological data indicated that pre-inoculation of barley with leaf rust increases the efficiency of the barley defense system to arrest the establishment of the challenger mildew fungus. This induction of resistance is localized rather than systemic. The lack of induction by the germinating snapdragon rust suggests that the effect is not caused by the rust germ tubes growing over the epidermis. The inducing effect by the rye leaf rust in barley suggests that rust haustoria are not essential either. We presume that the inducer effect is due to rust-plant communication during stoma penetration by the rust fungus, or to the presence of the rust hyphae in the apoplast, or to some degree of pre-haustorial resistance mounted by the mesophyll cells to the invading rust fungus. The level of induced mildew resistance varies among barley accessions. Although great differences in level were found among Pallas near-isogenic lines (Figure 2), suggesting a role of the Ml genes in these lines, the virulence or avirulence of the challenging mildew apparently did not matter (Figure 2A versus 2B). The high level of induced resistance observed in Pallas-Mla3 and Pallas-MlLa compared to Pallas may be due to some other genetic difference between these NILs either linked to the Ml gene, or elsewhere on the genome. Also we did not find sufficient evidence that level of basal resistance to P. hordei determines the level of induced resistance. It is intriguing that the experimental line 17-5-16 that shows a high level of basal resistance to P. hordei, shows also a high level of induced resistance. However, this may be a co-incidental association, since the two Vada NILs with a decreased level of basal resistance ( rphq2 ), did not induce a convincingly lower level of induced resistance than the two lines carrying the alleles Rphq2 for higher level of basal resistance (Figure 3). The control treatment inoculated only with the PM suggests that the lower the basal resistance to powdery mildew (Manchuria, Figure 2), the lower the induction of penetration resistance by the rust. This possible association should be verified in larger sets of barley accessions, for which we might use the material characterized in Chapter 2 and 3, showing a wide range of levels of such basal resistance. If indeed the level of rust induced resistance depends on the level of basal resistance to mildew, an interesting hypothesis would be that the rust “primes” the basal mildew resistance genes prior to the challenge mildew infection. Such a “priming” of defense genes (ISR) has been reported to occur in plants that were colonized by Pseudomonas fluorescence (Verhagen et al ., 2004).

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A Mildew LR LR+PM 4

1 Log signal intensity 0

se in a PR-1 bitor h i t rote yn s l p e inh a s e c in ti he ste t Protea y ogenesis-related p. C th Hypo Pa

B 4

1 Log signal intensity 0

. a p . rase nase b 0 kD d i b 1 y k / I h a . I ll t an y c ph Pho o or Ribulose-1,5-b. c. hl Carboni C

Phosphoglycerate Figure 6 Examples of highly up-regulated and down-regulated host genes in PM, LR and LR+PM treatments. A, five genes with the high DI and B, five genes with the low DI. Data represent mean of signal intensities of three inoculation experiments.

Macroarray transcriptome analysis To get insight into the molecular basis of the rust-induced mildew resistance phenomenon, we compared the global gene expression profile of double inoculated barley plants with that of single inoculated ones. We found a considerable level of overlap in differentially regulated genes in double inoculated plants with that of single powdery mildew or rust inoculated ones (25% and 67% respectively) although these two pathogens are adapted to live in different

Rustinduced powdery mildew resistance host tissues. P. Schweizer (IPK, Germany, unpublished data) found a considerable overlap in transcriptional response between entire leaf samples challenged with powdery mildew or rust, suggesting that rust and powdery mildew affect the barley transcriptome in a similar way. However, since powdery mildew interacts exclusively with the epidermis of the host, a considerable proportion of powdery mildew induced transcripts that are epidermis-specific may escape from detection in whole-leaf samples due to dilution of the signal by the extract of the mesophyll (Zierold et al. , 2005). Accordingly, this experiment was initially set up to compare gene expression using peeled epidermis. However, this turned out to be technically not feasible because rust pustules damaged the epidermis and thereby, prevented efficient peeling by 6 dai. Therefore only whole leaf samples were collected. By using DI as criterion for differential gene regulation we identified 46 genes belonging to different functional categories, predominantly activated in the double inoculated plants versus single powdery mildew inoculated plants. In double inoculated and also single rust inoculated plants, high up-regulation of PR-1 protein, a marker for induced resistance was observed. Pathogenesis-related proteins (PRs) are usually defined as host-specific proteins that are induced in several plant species in response to attack by pathogens or related situations and have antimicrobial activity (van Loon et al ., 2006). Transient silencing of PR-1 expression decreased the penetration resistance to the powdery mildew fungus in barley, indicating a role for PR-1 in basal resistance to mildew (Schultheiss et al ., 2003). We observed high up- regulation of Cysteine synthase (Table 2) in the double inoculated treatment but not in single rust or powdery mildew inoculated treatments. Fofana et al ., (2007) reported that in interaction of wheat with the wheat leaf rust pathogen ( Puccinia triticina ) the expression of Cysteine synthase is up-regulated in incompatible (Lr gene) but down- regulated in compatible interactions. Cysteine synthase is involved in reducing the concentration of reactive oxygen species (ROS), signaling molecules that are involved in various processes including pathogen defense (Apel & Hirt, 2004). However, to confirm that PRs and ROS are indeed involved in rust-induced mildew resistance, further investigations are necessary. Biotrophic fungal pathogens including rust and powdery mildews were reported to decrease the rate of photosynthesis in their host plants (e.g. Scholes & Farrar, 1986; Scholes et al ., 1994; Scholes & Rolfe, 1996). Our results showed that many genes that were strongly down-regulated in double inoculated plants are involved in photosynthesis (Table 2). Similar down-regulation of genes involved in photosynthesis reported in wheat attacked by leaf rust pathogen including the 1,5- Ribulose bisphosphate carboxylase which is the main source of energy production

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for the plant cell, generating ATP and reductive potential (NADPH) through photosynthesis (Fofana et al ., 2007). The role of this high down-regulation of genes involved in photosynthesis in rust-mildew induced resistance is unknown, but it is tempting to speculate that the strong suppression of photosynthesis in the mesophyll might prevent proper powdery mildew development in the epidermis due to the lack of energy. This study presents the detection and histological and molecular characterization of rust induced mildew resistance. Important questions that need to be addressed in subsequent studies are the genes that determine the level of induced resistance, and the mechanisms by which the rust infections apparently reprogramme the epidermal cells to become more resistant to the mildew pathogen. An entirely different type of question is, whether this interaction may have epidemiological consequences in case both pathogens occur side-by-side in the field.

Acknowledgement Reza Tadayonirad is acknowledged for his great help during the inoculations and samplings. The macroarray analysis was done at the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). The excellent technical assistance of Ines Walde is acknowledged. We thank Dr. Axel Himmelbach from IPK and Dr. Chris Maliepaard form Plant Breeding for thier help in data analysis.

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Gregersen PL, Smedegaard V. 1989. Induction of resistance in barley against Erysiphe graminis f.sp . hordei after preinoculation with the saprophytic fungus, Cladosporium macrocarpum. Journal of Phytopathology 124: 128–136 . Halperin T, Schuster S, PniniCohen S, Zilberstein A, Eyal Z. 1996 . The suppression of pycnidial production on wheat seedlings following sequential inoculation by isolates of Septoria tritici . Phytopathology 86: 728–732. Hammerschmidt R, Kuc J. 1982. Lignification as a mechanism for induced systemic resistance in cucumber. Physiological Plant Pathology 17: 61–71. Heath MC. 2002. Cellular interactions between biotrophic fungal pathogens and host and nonhost plants. Canadian Journal of Plant Pathology 24: 259–264. Holub EB, Cooper A. 2004. Matrix, reinvention in plants: how genetics is unveiling secrets of non- host disease resistance. Trends in Plant Science 9: 211-214. Hwang BK, Heitefuss R. 1982. Induced resistance of spring barley to Erysiphe graminis f.sp hordei . Phytopathol. Z . 103: 41–47. Jørgensen HJL, Lübeck PS, Thordal-Christensen H, de Neergaard E, Smedegaard-Petersen V. 1998. Mechanisms of induced resistance in barley against Drechslera teres . Phytopathology 88: 698-707. Kessler A,. Baldwin IT. 2002. Plant responses to insect herbivory: the emerging molecular analysis. Annual Review Plant Biology 53: 299–328. Kochman J, Brown J F. 1975. Studies on the mechanism of cross protection in cereal rusts. Physiological Plant Pathology 6: 19–27. Koga H, Bushnell WR, Zeyen RJ. 1990. Specificity of cell type and timing of events associated with papilla formation and the hypersensitive reaction in leaves of Hordeum vulgare attacked by Erysiphe graminis f. sp. hordei . Canadian Journal of Botany 68: 2344–2352.

Kogel KH, Langen G. 2005. Induced disease resistance and gene expression in cereals. Cell Microbiology 7: 1555–1564. Kovats K, Binder A, Hohl HL. 1991. Cytology of induced systemic resistance of tomato to Colletotrichum lagenarium . Planta 183: 484–490. Kunoh H, Katsuragawa N, Yamaoka N, Hayashimoto A. 1988. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. III. Timing and localization of enhanced inaccessibility in a single coleoptile cell and its transfer to an adjacent cell. Physiological and Molecular Plant Pathology 33: 81–93. Kunoh H. 2002. Localized induction of accessibility and inaccessibility by powdery mildew. In: Bélanger RR, Bushnell WR, Dik AJ, Carver TLW, eds. The powdery mildews: A comprehensive treatise. St. Paul/Minnesota, USA: APS Press, 126–133. Lin MR, Edwards HH. 1974. Primary penetration process in powdery mildewed barley related to host cell age, cell type, and occurrence of basic staining material. New Phytologist 73: 131-137. Lyngkjær MF, Carver TLW, Zeyen RJ. 2001. Virulent Blumeria graminis infection induces penetration susceptibility and suppresses race-specific hypersensitive resistance against avirulent attack in Mla1 -barley. Physiological and Molecular Plant Pathology 59: 243-256. Lyngkjær MF, Carver TLW. 1999. Induced accessibility and inaccessibility to Blumeria graminis f.sp. hordei in barley epidermal cells attacked by a compatible isolate. Physiological and Molecular Plant Pathology 55: 151–162.

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Lyngkjær MF, Carver TLW. 2000 . Conditioning of cellular defence responses to powdery mildew in cereal leaves by prior attack. Molecular Plant Pathology 1: 41-50. Mains EB, Dietz SM. 1930. Physiologic forms of barley mildew, Erysiphe graminis hordei Marchal. Phytopathology 20: 229-39. Manandhar HK, Jørgensen HJL, Mathur SB, Smedegaard- Petersen V. 1998. Suppression of rice blast by preinoculation with avirulent Pyricularia oryzae and the nonrice pathogen Bipolaris sorokiniana . Phytopathology 88: 735-739. Marcel TC, Aghnoum R, Durand J, Varshney RK, Niks RE. 2007. Dissection of the barley 2L1.0 region carrying the ‘Laevigatum’ quantitative resistance gene to leaf rust using near isogenic lines (NIL) and subNIL. Molecular Plant–Microbe Interactions 20: 1604–1615. Mellersh DG, Heath MC. 2001. Plasma membrane – cell wall adhesion is required for expression of plant defense responses during fungal penetration. Plant Cell 13: 413–424. Nelson HE. 2005. Fusarium oxysporum f.sp. radicis-lycopersici can induce systemic resistance in barley against powdery mildew. Journal of Phytopathology 153: 366-370. Niks RE. 1986. Failure of haustorial development as a factor in slow growth and development of Puccinia hordei in partially resistant barley seedlings. Physiological and Molecular Plant Pathology 28: 309–322. Niks RE. 1989. Induced accessibility and inaccessibility of barley cells in seedling leaves inoculated with two leaf rust species. Journal of Phytopathology 124: 296–308. Oku H, Ouchi S. 1976. Host plant accessibility to pathogens. Review of Plant Protection Research 9: 58–71. Olesen KL, Carver TLW, Lyngkjær MF . 2003. Fungal suppression of resistance against inappropriate Blumeria graminis formae speciales in barley, oat and wheat. Physiological and Molecular Plant Pathology 62: 37-50. Oostendorp M, Kunz W, Dietrich B, Staub T. 2001. Induced disease resistance in plants by chemicals. European Journal of Plant Pathology 107: 19–28. Ouchi S, Hibino C, Oku H. 1976a. Effect of earlier inoculation on the establishment of a subsequent fungus as demonstrated in powdery mildew of barley by a triple inoculation procedure. Physiological and Molecular Plant Pathology 9: 25–32. Ouchi S, Oku H, Hibino C. 1976b. Localization of induced resistance and susceptibility in barley leaves inoculated with the powdery mildew fungus. Phytopathology 66: 901-905. Parlevliet JE, Kuiper HJ. 1985. Accumulating polygenes for partial resistance to barley leaf rust, Puccinia hordei . 1. Selection for increased latent period. Euphytica 37: 7–13. Pei MH, Ruiz C, Hunter T, Bayon C. 2003. Rust resistance in Salix induced by inoculations with avirulent and virulent isolates of Melampsora larici-epitea . Forest Pathology 33: 383–94. Peterhänsel C, Freialdenhoven A, Kurth J, Kolsch R, Schulze-Lefert P. 1997. Interaction analyses of genes required for resistance responses to powdery mildew in barley reveal distinct pathways leading to leaf cell death. Plant Cell 9: 1397–1409. Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD. 1996. Systemic acquired resistance, Plant Cell 8: 1809–1819. Sahashi N, Shishiyama J. 1986. Increased papilla formation, a major factor of induced resistance in the barley-Erysiphe graminis f.sp . hordei system. Canadian Journal of Botany 64: 2178-2181. Scholes JD, Farrar JF.1986. Increased rates of photosynthesis in localised regions of a barley leaf infected with brown rust. New Phytologist 104: 601–612.

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Scholes JD, Lee PJ, Horton P, Lewis DH. 1994 . Invertase: understanding changes in the photosynthetic and carbohydrate metabolism of barley leaves infected with powdery mildew. New Phytologist 126: 213-222. Scholes JD, Rolfe SA. 1996. Photosynthesis in localised regions of oat leaves infected with crown rust ( Puccinia coronata ): quantitative imaging of chlorophyll fluorescence . Planta 199: 573-582. Schultheiss H, Dechert C, Király L, Fodor J, Michel K, Kogel K-H, Huckelhoven R. 2003. Functional assessment of the patgogenesis-related protein PR-1b in barley. Plant Science 165: 1275–1280. Schweizer P, Hunziker, Mosinger E. 1989. cDNA cloning, in vitro transcription and partia1 sequence analysis of mRNAs from winter wheat (Triticum aestivum L.) with induced resistance to Erysiphe graminins f.sp. tritici. Plant Mo1ecular Biology 12: 643-654. Sebesta J, Epstein AH, Formanova M. 1996. Induction to resistance to crown rust in oat. Journal of phytopathology 144: 455-458. Sreenivasulu N, Altschmied L, Panitz R, Hahnel U, Michalek W, Weschke W, Wobus U. 2002. Identification of genes specifically expressed in maternal and filial tissues of barley caryopses: a cDNA array analysis. Molecular Genetics and Genomics 266: 758–767. Sticher L, Mauch-Mari B, Metraux JP. 1997. Systemic acquired resistance. Annual Review of Phytopathology 35: 235–270. Ton J, Pieterse CMJ, Van Loon LC. 2006. The relationship between basal and induced resistance in Arabidopsis . In: Tuzun S, Bent E, eds. Multigenic and induced systemic resistance in plants . New York, USA: Springer, 197–224. Trujillo M, Troeger M, Niks RE, Kogel KH, Hückelhoven R. 2004. Mechanistic and genetic overlap of barley host and non-host resistance to Blumeria graminis . Molecular Plant Pathology 5: 389- 396. Tsuchiya K, Hirata K. 1973. Growth of various powdery mildew fungi on the barley leaves infected preliminarily with the barley powdery mildew fungus. Annals of the Phytopathological Society of Japan 39: 396–403. Vallad GE, Goodman RM. 2004. Systemic acquired resistance and induced systemic resistance in conventional agriculture. Crop Science 44: 1920-1934. Vallelian BL, Mosinger E, Metraux JP, Schweizer P. 1998. Structure, expression and localization of a germin-like protein in barley Hordeum vulgare L. that is insolubilized in stressed leaves. Plant Molecular Biology 37: 297–308. Van Loon LC, Bakker PAHM, Pieterse CMJ. 1998. Systemic resistance induced by rhizosphere bacteria. Annual Review of Phytopathology 36: 453–483. van Loon, LC, Rep M, Pieterse CMJ. 2006. Significance of inducible defense related proteins in infected plants. Annual Review of Phytopathology 44 : 1-28. Verhagen BWM, Glazebrook J, Zhu T, Chang H, Van Loon LC, Pieterse CMJ. 2004 . The transcriptome of rhizobacteria-induced systemic resistance in Arabidopsis. Molecular Plant- Microbe Interaction 17: 895-908. Yarwood CE. 1954. Mechanism of acquired immunity to a plant rust. Proceedings of the National Academy of Sciences of the United States of America 40: 374-377. Zierold U, Scholz U, Schweizer P. 2005. Transcriptome analysis of mlo -mediated resistance in the epidermis of barley. Molecular Plant Pathology 6: 139-152.

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Introduction In the barley-Blumeria interaction attacked plant epidermal cells defend themselves against fungal penetration by localized responses leading to papilla formation and preventing the fungus from haustorium formation. This mechanism of defense conveys a race non-specific form of resistance and is common to basal host resistance and mlo -mediated resistance to adapted powdery mildew ( Bgh ) and non- host resistance to non-adapted forms of Bg. Identification of molecular components underlying mlo resistance in barley and non-host resistance in Arabidopsis revealed that these two types of resistance share many similarites (Humphry et al ., 2006). However, it is not known which genes make some barley accessions less susceptible to the adapted form of Bg than others, and which genes determine the host status of barley to non-adapted Bg forms. We undertook an integrated approach combining QTL mapping and candidate genes analysis in order to identify genes that are involved in basal resistance to adapted Bgh . Two experimental barley lines were developed with exceptional susceptibility to a non-adapted form of Bg , the wheat powdery mildew fungus . In this Chapter we discuss the implications of our findings for understanding the genetics and mechanisms of basal resistance of barley to adapted and non-adapted forms of powdery mildew.

Abundance and diversity of genes for basal resistance to powdery mildew Basal resistance of barley to powdery mildew is a quantitatively inherited trait that limits the growth and sporulation of powdery mildew colonies by a non-hypersensitive mechanism of defense. This resistance is valuable for the breeders since it is assumed to be race non-specific and durable. However, due to the polygenic inheritance, interaction with environmental factors and subjectivity of disease assessment, this type of resistance has always been considered difficult to breed for. New molecular tools provided the opportunity to dissect this type of resistance and understand its molecular basis, and might lead to more efficient breeding methods. QTL mapping has broaden our understanding of the number and location of the genes that are involved in basal resistance in different genetic backgrounds of barley (Chapter 2). Most mapping studies on basal resistance of barley to powdery mildew have been based on single biparental mapping populations, without verfication of the mechanism of resistance at the cellular level. QTL mapping in biparental populations reveals only a slice of the genetic architecture for a trait because only alleles that differ between the two parental lines will segregate (Holland, 2007). In this thesis, the genetics and mechanism of basal resistance of barley to powdery mildew have been

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Co-localization of QTLs and candidate genes Gene expression studies, motif directed profiling and high throughput gene silencing, transient transformation and large scale mutation assays have been very productive to indicate candidate genes for traits of interest, including disease resistance. In Chapter 2, we prioritized many candidate genes that showed an interaction phenotype with powdery mildew after RNAi silencing or overexpression studies, by acquiring evidence of their mapping position. Six out of 26 mapped candidate genes co-localized with the QTLs for powdery mildew resistance. This co-localization suggests that those candidate genes are likely to contribute to the variation observed in the mapping population in which the QTL at that locus had been detected. The information obtained from the co-localization and the knowledge of the sequences of those candidate genes can speed up the isolation of the genes at those QTLs. However, for final proof that the QTLs that co-localize with the candidate genes are indeed caused by allelic variation in those candidate genes, a mapping at much higher genetic resolution is required. HvSNAP34 is one of the prioritized candidate genes. This gene encodes a t-SNARE protein that is involved in mlo -mediated, non- host and host basal resistance (Douchkov et al ., 2005). This is an interesting gene to

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be cloned to compare its allelic variation between Dom and Rec, that had different QTL alleles at that locus. Both alleles could be used in transient expression assays to investigate whether one confers more resistance than the other.

Accumulation of genes for basal resistance The multi-population QTL analysis showed that basal resistance of barley to powdery mildew is conferred by many genes, most of which are growth stage dependent. QTL analysis followed by Marker Assisted Selection (MAS) has been suggested to be an efficient strategy in developing new lines with levels of resistance superior to that of their parents. This process is known as QTL pyramiding. For pyramiding, after the discovery of interesting QTLs in the mapping population, QTLs can be combined through crossing of the genotypes carrying the QTL alleles to be followed by MAS (Ashikari & Matsuoka, 2006). However in practical breeding, accumulation of genes using MAS has to be repeated in each crossing cycle since the combined resistance genes will segregate in the progeny. For traits that are difficult to measure and show a low heritability, the MAS strategy should be considered as a potential alternative for crop improvement. However, there are not always marker alleles close to the desired QTL allele to monitor reliably the introgression of that QTL in new progeny. Other parents may carry the same marker allele, or the marker allele may be too far away from the target QTL allele. In that case haplotypes around the target QTL allele should be determined, which is a laborious effort. Another aspect that may reduce the efficiency of MAS is the possible occurrence of QTL interactions. That may lead to disappointing phenotypic performance conferred by designed QTL combinations. We showed here that accumulation of unknown QTLs just by visual selection for higher level of basal resistance among the progenies is feasible and easy.

Non-host immunity of barley to non-adapted powdery mildews Complexity of the mechanism of non-host resistance Many hypotheses about the mechanisms of non-host resistance have been suggested, but two of them are currently in the focus of interest. 1-The non-adapted pathogens produces effectors to suppress PAMP-triggered immunity in the plant, but these effectors correspond insufficiently with the operative targets. 2- Plants contain multiple “ R genes” acting together to recognize the pathogen avirulence (Avr) gene products (functionally these are presumed to be effectors). Since the multiple R-gene combination requires several simultaneous Avr avr mutations, it is impossible for the pathogen to break this defense.

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Both of these two models can explain the durability of non-host resistance (Jones & Dangl, 2006). Genetic studies of interaction of Arabidopsis with non-adapted powdery mildew forms have shown that both pre- and post-penetration defense mechanisms are involved in non-host resistance (Lipka et al ., 2005). Our histological studies revealed that resistance to penetration in association with papilla formation is the main mechanism involved in non-host resistance to non-adapted forms of Bg. However, a considerable proportion of successfully penetrated germlings (30 to 60% depending on the barley-non-adapted mildew form combination) were stopped by a hypersensitive reaction. One forma specialis , from Hordeum murinum , induced extensive hypersensitivity upon haustorium formation on some tested lines. However, Christopher-Kozjan & Heath, (2003) presented evidence that hypersensitivity in non- host combinations not necessarily imply the activity of R/Avr interaction. The combination of non-hypersensitive and hypersensitive defense mechanisms involved suggest that non-adapted powdery mildew forms should evolve strategies to overcome different defense mechanisms in different developmental stages to acuire a plant species as suitable host.

Genetics of non-host resistance to non-adapted powdery mildews Most of the genes that have been implicated in non-host resistance have been discovered by mutagenesis or gene expression studies (Niks & Marcel, 2009; Table 1). Only a few studies reported genes that play a role in natural variation for non-host resistance (Shafiei et al. , 2007; Rodrigues et al ., 2004; Jafary et al ., 2006; Jafary et al ., 2008; Zhang et al ., 2009). Barley is a full non-host to the non-adapted Bg t. We tested here a large collection of barley accessions against Bgt, and only a few accessions were found to possess susceptibility to Bg t at seedling stage, and at most at a very low level. Crossings among some barley accessions with some level of susceptibility to Bg t resulted in the accumulation of susceptibility factors in two barley lines called SusBgt SC and SusBgt DC. The accumulation of susceptibility genes in barley to Bgt by successive crosses suggest that the resistance has a quantitative inheritance. However we intend to follow a mapping approach to determine the genetics of non-host resistance in barley against non-adapted forms of Bg. Furthermore both SusBgt lines are useful tools in gene expression and complementation studies on non-host resistance. The two lines show surprisingly a differential level of susceptibility in the conidiation stage of non-adapted powdery mildews. Therefore, mapping populations based on both lines may lead to the identification of QTLs that are effective in particular stages of fungal development.

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Enhancement of basal resistance to powdery mildew Our results showed that basal resistance of barley against powdery mildew is enhanced by pre-inoculation with leaf rust. We hypothesized that this rust-induced mildew resistance might be due to rust-plant communication during stoma penetration, or to the presence of the rust hyphae in the apoplast, or to some degree of pre-haustorial resistance mounted by the mesophyll cells to the invading rust fungus. Using a barley cDNA macroarray, we compared the genome-wide transcriptome changes upon double inoculation (LR+PM) with single inoculation leaf rust (LR) and powdery mildew (PM) treatments. The majority of genes up-regulated by LR are also up-regulated in the LR+PM, and hence, not silenced by PM. Analysis of quantitative transcriptome abundance of differentially regulated genes in double inoculation treatment versus single inoculations (PM and LR) revealed that most of the down- regulated genes are involved in plant metabolism and photosynthesis. This suggests that the rust-induced powdery mildew resistance state might be due to a lack of energy in the plant cells. In double inoculated and also single rust inoculated plants, a high up-regulation of PR-1 protein, a Pathogenesis-related protein (PR) was observed. Accumulation of PRs is a hallmark of pathogen-induced systemic acquired resistance (van Loon et al ., 2006). Accumulation of PR proteins after infection by pathogens has been reported to be associated with increased effectiveness of plant defense responses upon subsequent pathogen infection, a state that has been called ‘’Priming’’. Primed plants are capable of activating defense responses either faster, stronger, or both upon attack by pathogens, insects or in response to abiotic stress (Conrath et al., 2006). Both our histological and expression studies suggest that pre- inoculation of barley with leaf rust prime the basal defense mechanism of plants against subsequent challenge inoculation with powdery mildew.

Concluding remarks In this thesis we found that the barley genome is a rich source of QTLs (genes) conferring basal resistance to powdery mildew. The great diversity of such genes in the barley germplasm making transgression for basal resistance a very common phenomenon. Identification of the genes underlying the powdery mildew seems feasible considering the prioritarized candidate genes that were found to co-localize with resistance QTLs. Information gained from the prioritization of candidate genes can help in the isolation of the genes that underlie resistance QTLs. These candidate genes have been shown already to affect the level of basal resistance, and now they have been mapped to the QTL positions found in our mapping studies. In qualitative

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Table 1. Genes with known sequences that were implicated to play a role in basal host and/or non- host resistance of barley to powdery mildews ( adapted from Niks & Marcel, 2009)

Gene name Description BHR a NHR b References *

Genes involved in transport and secretion systems ROR2 Plasma membrane-anchored Bgh Bgt 1,2,3,8,10 syntaxin SNAP34 Synaptosome-associated protein Bgh Bgt 11,3,10 ADF3 Actin depoly merizing factor 3 Bgh Bgt 11

Genes involved in the positive or negative regulation of basal defenses BI-1 BAX inhibitor 1 Bgh Bgt 3,9,11 CDPKs Calcium-dependent protein Bgh Bgt 8 kinases MLO Seven-transmembrane domain Bgh 2,3,4,7,10,11 protein

Genes involved in redox signaling GLPs Germin-like proteins Bgh 2,3 RBOHs NADPH oxidase Bgh 3 PRXs: Prx07/Prx40 Class III peroxidase proteins Bgh Bgt 6

Transcription factors and signaling cascades NACs: NAC6 NAC transcription factors Bgh 5 WRKYs: WRKY1/2 Group II WRKYs transcription Bgh 12 factors a, basal host resistance b, non-host resistance * references : 1: Collins et al. (2003) 5 : Jensen et al. , (2007) 9: Nürnberger & Lipka (2005) 2: Collins et al. (2007) 6: Johrde & Schweizer (2008) 10: O’Connell & Panstruga (2006) 3: Hückelhoven (2007) 7: Kumar et al. (2001) 11: Schweizer (2007) 4: Jarosch et al. (1999) 8: Lipka et al. (2008) 12: Shen et al. (2007)

genes this would be sufficient proof to consider these sequences as the “responsible gene”. In QTLs it is, however, too early to consider these candidates as “confirmed”. We propose that either complementation tests with allelic forms or ultra-fine physical QTL mapping are carried out to confirm the candidate genes that emerge from our study as the most likely. The developed barley lines with exceptional susceptibility to Bgt will serve to determine the genetic basis of non-host resistance in barley to wheat powdery mildew by both forward and reverse genetic approaches. This material will also be useful to quantify subsequent lines of basal defense that the mildew fungus encounters during its development, such as primary haustorium and secondary haustorium formation and conidiation. Finally, the rust-induced mildew resistance

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phenomenon that was reported here for the first time can be further investigated to improve our knowledge about the molecular mechanism of penetration resistance at cell level and the mechanisms that plants deploy to protect themselves to different biotic stress agents.

References Ashikari M, Matsuoka M. 2006. Identification, isolation and pyramiding of quantitative trait loci for rice breeding. Trends Plant Science 11: 344–350. Christopher-Kozjan R, Heath MC. 2003. Cytological and pharmacological evidence that biotrophic fungi trigger different cell death execution processes in host and nonhost cells during the hypersensitive response. Physiological and Molecular Plant Patholology 62: 265-275. Collins NC, Niks RE, Schulze-Lefert P, 2007. Resistance to cereal rusts at the plant cell wall-What can we learn from other host-pathogen systems? Australian Journal of Agricultural Research 58: 476-489. Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu J-L, Hückelhoven R, Stein M, Freialdenhoven A, Somerville S et al. 2003. SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425: 973-977. Conrath U, Beckers GJM, FlorsV, Garcia-Agustin P, Jakab G, Mauch F, Newman MA, Pieterse CMJ, Poinssot B, Pozo MJ, Pugin A, Schaffrath U, Ton J, Wendehenne D, Zimmerli L, Mauch-Mani B. 2006. Priming: getting ready for battle. Molecular Plant-Microbe Interaction 19: 1062-1071. Douchkov D, Nowara D, Zierold U, Schweizer P. 2005. A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. Molecular Plant- Microbe Interactions 18: 755-761. Holland JB. 2007. Genetic architecture of complex traits in plants. Current Opinion in Plant Biology 10: 156–161. Hückelhoven R. 2007. Cell wall-associated mechanisms of disease resistance and susceptibility. Annual Review of Phytopathology 45: 101-127. Humphry M, Consonni C, Panstruga R. 2006. mlo -based powdery mildew immunity: silver bullet or simply non-host resistance? Molecular Plant Pathology 7: 605–610. Jafary H, Albertazzi G, Marcel TC, Niks RE. 2008. High diversity of genes for nonhost resistance of barley to heterologous rust fungi. Genetics 178: 2327–2339. Jafary H, Szabo LJ, Niks RE. 2006. Innate nonhost immunity in barley to different heterologous rust fungi is controlled by sets of resistance genes with different and overlapping specificities. Molecular Plant–Microbe Interactions 19: 1270–1279. Jarosch B, Kogel KH, Schaffrath U. 1999. The ambivalence of the barley Mlo locus: Mutations conferring resistance against powdery mildew ( Blumeria graminis f.sp. hordei ) enhance susceptibility to the rice blast fungus Magnaporthe grisea . Molecular Plant-Microbe Interactions 12: 508–514. Jensen MK, Rung JH, Gregersen PL, Gjetting T, Fuglsang AT, Hansen M, Joehnk N, Lyngkjaer MF, Collinge DB. 2007. The Hv NAC6 transcription factor: a positive regulator of penetration resistance in barley and Arabidopsis. Plant Molecular Biology 65: 137-150. Johrde A, Schweizer P. 2008. A class III peroxidase specifically expressed in pathogen-attacked barley epidermis contributes to basal resistance. Molecular Plant Pathology 9: 687- 696.

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General discussion

Jones JDG, Dangl JL. 2006. The plant immune system. Nature 444: 323–329. Kumar J, Hückelhoven R, Beckhove U, Nagarajan S, Kogel KH. 2001. A compromised Mlo pathway affects the response of barley to the necrotrophic fungus Bipolaris sorokiniana (Teleomorph: Cochliobolus sativus ) and its toxins. Phytopathology 91: 127–133. Lipka U, Fuchs R, Lipka V. 2008. Arabidopsis non-host resistance to powdery mildews. Current Opinion in Plant Biology 11: 1-8. Lipka V, Dittgen J, Bednarek P, Bhat R, Wiermer M, Stein M,Landtag J, Brandt W, Rosahl S, Scheel D, et al. 2005. Pre- and post invasion defences both contribute to nonhost resistance in Arabidopsis. Science 310: 1180-1183. Niks RE, Marcel TC. 2009. Non-host and basal resistance: how to explain specificity? New Phytologist 182: 817–828 Nürnberger T, Lipka V. 2005. Non-host resistance in plants: new insights into an old phenomenon. Molecular Plant Pathology 6: 335-345. O'Connell RJ, Panstruga R. 2006. Tête à tête inside the plant cell: establishing compatibility between plants and biotrophic fungi and oomycetes. New Phytolologist 171: 699-718. Poland JA, Balint-Kurti2 PJ, Randall J, Wisser RCP, Nelson RJ.2008 . Shades of gray: the world of quantitative disease resistance. Trends in Plant Science doi:10.1016/j.tplants.2008.10.006 Rodrigues P, Garrood JM, Shen QH, Smith PH, Boyd LA. 2004. The genetics of non-host disease resistance in wheat to barley yellow rust. Theoretical and Applied Genetics 109: 425-432. Schweizer P. 2007. Nonhost resistance of plants to powdery mildew - New opportunities to unravel the mystery. Physiological and Molecular Plant Patholology 70: 3-7. Shafiei R, Hang C, Kang J-G, Loake GJ. 2007. Identification of loci controlling non-host disease resistance in Arabidopsis against the leaf rust pathogen Puccinia triticina . Molecular Plant Pathology 8: 773-784. Shen, Q-H, Saijo Y, Mauch S, Biskup C, Bieri S, Keller B, Seki H, Ülker B, Somssich IE, Schulze- Lefert P. 2007. Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses. Science 315: 1098-1103. van Loon, LC, Rep M, Pieterse CMJ. 2006. Significance of inducible defense related proteins in infected plants. Annual Review of Phytopathology 44: 1-28. Zhang NW, Lindhout P, Niks RE, Jeuken MJW. 2009. Genetic dissection of Lactuca saligna nonhost resistance to downy mildew at various lettuce developmental stages. Plant Pathology , in press

109 Chapter 6

110 Abbreviations

Abbreviations AUDPC Area Under the Disease-Progress Curve Bg Blumeria graminis Bga Blumeria graminis f.sp. avenea Bgar Blumeria graminis f.sp. agropyri Bgb Blumeria graminis f.sp. bromi Bgd Blumeria graminis f.sp . dactylidis Bgh Blumeria graminis f.sp. hordei Bghm Blumeria graminis f.sp hordei-murini Bgt Blumeria graminis f.sp. tritici CAPS Cleaved Amplified Polymorphic Sequences dai day after inoculation DArT Diversity Arrays Technology DC Double Cross dCAPS derived Cleaved Amplified Polymorphic Sequences DHs Doubled Haploids DI Differential Index ESH Elongated Secondary Hypha EST Expressed Sequence Tag f.sp. forma specialis ff.spp. formae speciales FPP Failed Primary Penetration FPP+HR Failed Primary Penetration with HR FPP-HR Failed Primary Penetration without HR hai hour after inoculation HR Hypersensitive Response IR Induced Resistance IS Induced Susceptibility ISR Induced Systemic Resistance LOD Logarithm of odds LR Leaf Rust

111 Abbreviations

MAS Marker-Assisted Selection Mla Mildew resistance locus a Mlo Mildew resistance locus o nhR non-host resistance NILs Near Isogenic Lines PAMP Pathogen-Associated Molecular Patterns PEN Penetration (mutant genes) PM Powdery mildew PRs Pathogenesis-Related proteins PTI PAMP-Triggered Immunity QTL Quantitative Trait Locus Rbgq quantitative genes for Resistance to Blumeria graminis RILs Recombinant Inbred Lines RNAi RNA interference Ror Required for mlo -specified resistance Rphq2 An allele for quantitative resistance to Puccinia hordei SAP Secondary Attempted Penetration SAR Systemic Acquired Resistance SC Single Cross SCAR Sequence Characterized Amplified Region SPP Successful Primary Penetration SPP+HR Successful Primary Penetration with a haustorium and with HR SPP-HR Successful Primary Penetration with a haustorium but without HR TDM Transcript-Derived Marker TIGS Transient-Induced Gene Silencing

112 Glossary

Glossary Avirulent: carrying an avirulence (Avr) gene that hampers development and reproduction of pathogen in a host plant accession that carries a corresponding resistance gene. Basal defense/resistance: qualitative: defense that plant species mount against non-adapted microbial intruders. quantitative: defense that inhibits pathogen spread after successful infection and onset of disease. Basic compatibility: state that results from the capacity of the microbe to deal effectively with the defense that plant species mount against non-adapted microbial intruders. Challenger: in induced-resistance terminology, it refers to an adapted or non- adapted pathogen which is inoculated to a plant subsequently after inoculation with inducer pathogen (see there). Colonization: the development stage after establishment of the infection (see there). In mildews the stage of development of secondary and subsequent haustoria. Compatible interaction: a host–pathogen interaction that results in production of the pathogen without elicitation of gross defense responses (the host is susceptible). Durable resistance: resistance that has remained effective for a long period in which it has been applied at large scale in an environment conducive to the pathogen. Effector: secreted pathogen protein that manipulates host cell functions to suppress defense and promote infection. As a secondary effect, an effector may elicit in particular plant genotypes a hypersensitive response, and be an avirulence factor. Establishment: the development of a primary haustorium in attacked plant cells by a pathogenic fungus, especially rust and powdery mildew fungi. Haustorium: specialized organ formed by most biotrophic (pathogenic) fungi and oomycetes in a living plant cell for nutrient absorption and molecular communication with the plant cell. Host specificity: the property of a pathogen species to be able to establish basic compatibility with only one or a few plant species. Host status: status of a plant species being susceptible to a particular pathogen species. If not, the plant is a marginal host or a non-host. Incompatible interaction: a host–pathogen interaction that does not result in disease (the host is resistant).

113 Glossary

Induced Systemic Resistance (ISR): the phenomenon that plants acquire an enhanced defensive capacity against subsequent pathogen attack as a result of root colonization by selected strains of non-pathogenic bacteria. Inducer: In induced-resistance terminology, it refers to a pathogen, non-pathogenic microorganism or other factors that induce resistance or susceptibility to a subsequent challenger. Linkage disequilibrium (LD). The condition in which the frequency of a particular haplotype for two loci is significantly different from that expected under random mating. Near isogenic lines (NILs): Inbred lines that differ at only a small genomic region. Non-adapted pathogen: pathogen species that cannot infect a particular (non-host) plant species (syn. heterologous, inappropriate). Non-host resistance: resistance occurring in all genotypes of a plant species to all genotypes of a potential pathogen species. Operative target: host target that, when manipulated by a pathogen effector, results in enhanced pathogen fitness. PAMP: pathogen-associated molecular pattern; a pathogen-specific molecular sequence or structure, often indispensable for the microbial lifestyle that, elicits an innate immune response on receptor-mediated perception. PAMP-triggered immunity (PTI): first line of active plant defense that relies on the recognition of pathogen-associated (or microbe-associated) molecular patterns (PAMPs/MAMPs) by pattern-recognition receptors (PRRs). Pattern-recognition receptors: plant proteins that identify PAMP (see above) molecules, such as flagellin or chitin components, that are associated with microbial pathogens. Quantitative trait locus (QTL): a locus with an effect on a quantitative trait (i.e. a trait showing continuous variation) that can only be mapped by the application of QTL mapping software. Recombinant inbred line (RIL): an inbred line produced from an initial cross followed by continuous inbreeding; populations of RILs are often used for QTL mapping studies. Resistance gene analogs (RGA): a sequence identified in whole genome analyses based on their structural homology (presence of NBS and/or LRR domains) with previously identified R-genes. Systemic Acquired Resistance (SAR): the phenomenon that plants acquire an enhanced defensive capacity against subsequent pathogen attack as a result of pre- inoculation with pathogens that causes necrotic lesions.

114 Summary

Summary In the barley-Blumeria interaction, resistance at penetration stage in association with papilla formation is a commonly occurring mechanism. This mechanism of defense reduces the infection severity by adapted powdery mildew pathogen (basal resistance to Blumeria graminis f.sp. hordei , Bgh ) and fully protects the plant against non-adapted powdery mildew pathogens (non-host resistance to non-adapted forms of B. graminis ). In this thesis we followed an integrated approach based on QTL mapping and candidate gene analysis. It was our objective to determine which of the known candidate genes have a map position that coincides with one of the QTLs detected in our mapping study. Such candidate genes might determine the natural variation of basal resistance against powdery mildew in barley. We also investigated whether the genotypic variation in level of resistance of barley to the non-adapted wheat powdery mildew, B. graminis f.sp. tritici (Bgt ) could be used to develop an experimental line to determine the inheritance of non-host resistance of barley to this pathogen. Finally we aimed to determine whether the basal resistance of barley to powdery mildew is being suppressed by prior attack by a different pathogenic haustorium forming fungus, Puccinia hordei . Chapter 1 presents an introduction about basal resistance of barley to powdery mildew and briefly lists evidence on molecular associations between basal, mlo - mediated and non-host resistance. In Chapter 2 , we performed QTL analysis for powdery mildew resistance at seedling and at adult plant stage in six mapping populations of barley. In that analysis quantitative resistance of barley to powdery mildew was found to be based on a large number of genes, with only few detected in more than one mapping population. These QTLs for powdery mildew resistance in barley are mainly plant growth stage dependent. From gene-expression, gene silencing and transient transformation assays 39 genes emerged that increased or decreased basal resistance of barley to the powdery mildew fungus. They are considered candidate genes that might be responsible for natural variation in level of basal resistance. By looking for polymorphism in those genes, we could determine the map position of 23 of these candidate genes. Mapping positions of three more genes, mlo , Ror1 and Ror2, were already available, and these were also considered as candidate genes in our study.To compare the map position of QTLs in different mapping populations with the map positions of the 26 candidate genes, we constructed an improved high-density integrated linkage map of barley. This improved map was based on the marker data of seven mapping populations of barley. More than 43% of the markers on our improved integrated map target ESTs or gene sequences (gene-targeted markers, GTM) and can be used for candidate

115 Sammary

gene identification. Six out of the 26 candidate genes co-localized with QTLs for basal powdery mildew resistance. They are interesting targets for further studies, since their allelic forms may be responsible for part of the differences in basal resistance between the parents in our mapping study. Chapter 3 reports the development of two experimental barley lines with extremely high and low level of basal resistance to barley powdery mildew. These lines were obtained by convergent crossing between the most resistant and the most susceptible lines, respectively, from four mapping populations of barley studied in Chapter 2. We consider the extremely susceptible and resistant lines developed here as valuable material to be used in further experiments to characterize the molecular basis of basal resistance to powdery mildew. The results suggest that phenotypic selection is sufficiently efficient to achieve high levels of basal resistance. We report some difficulties in the application of marker assisted selection that might make that approach unnecessary and less efficient to achieve a similar high level of basal resistance. In Chapter 4 the development of two more experimental lines is described. These lines have, at the seedling stage an unprecedented level of susceptibility to the non-adapted wheat powdery mildew. A large collection of barley germplasm was screened and some rare barley accessions were identified with rudimentary susceptibility to the wheat powdery mildew, Bgt . Those accessions were intercrossed in two cycles, and resulted in the two exceptional research lines, called SusBgt SC and SusBgt DC . The quantitative variation among barley accessions and in the progenies after convergent crossing suggest a polygenic basis of this non-host resistance. Component analysis of the infection process suggested that non-host resistance factors are Blumeria -form specific. The developed lines will serve to elucidate the genetic basis of non-host resistance in barley to wheat powdery mildew, and are useful tools in gene expression and complementation studies on non-host resistance. In Chapter 5 we reported that a compatible interaction of barley-Puccinia hordei induces increased papilla based resistance to a challenge infection by a compatible powdery mildew isolate. These pathogens differ in strategies to suppress plant cell defense. We showed that the level of rust-induced mildew resistance varies among barley accessions and is not determined by the virulent/avirulent spectra of the challenger isolate. Macroarray gene expression analysis showed that several genes involved in metabolism and photosynthesis were highly down-regulated and a few genes involved in plant defense including a pathogenesis related protein (PR-1) and cysteine synthase were highly up-regulated in the double inoculated treatment. Our histological and gene expression analyses are compatible with the hypothesis that the rust “primes” the basal mildew resistance genes prior to the challenge mildew

116 Summary

infection. In Chapter 6 , the results obtained in the previous Chapters are being discussed. The perspectives of the experimental barley lines that were developed in this study for the identification of genes that determine basal resistance of barley to adapted and non-adapted Blumeria graminis forms are highlighted.

117 Sammary

118 Samenvatting

Samenvatting In de gerst-meeldauw ( Blumeria ) interactie is resistentie tegen celwandpenetratie door de vorming van papillen een algemeen voorkomend verschijnsel. Dit resistentiemechanisme reduceert de aantastingsgraad van gerst door gerstmeeldauw (basisresistentie tegen Blumeria graminis f.sp. hordei , Bgh ), en beschermt de plant volledig tegen niet op gerst gespecialiseerde vormen van meeldauw (niet-waardresistentie). In dit proefschrift volgden we een geïntegreerde benadering door de kartering van QTLs te combineren met gegevens uit een kandidaatgenbenadering. Doel was om te bepalen welke van de bekende kandidaatgenen karteren op een van de door ons in kaart gebrachte QTLs. Dergelijke kandidaatgenen kunnen verantwoordelijk zijn voor de gevonden verschillen in basisresistentie van gerst tegen de gerstmeeldauw. We hebben ook onderzocht of de genetische variatie in resistentieniveau tegen de tarwemeeldauwschimmel ( B. graminis f.sp. tritici ) zou kunnen worden benut om een experimentele gerstlijn te ontwikkelen die zou kunnen dienen om de overerving te bepalen van de niet-waard resistentie van gerst tegen dat pathogeen. Tenslotte bepaalden we of de basisresistentie van gerst tegen meeldauw zou worden onderdrukt na eerdere infectie door een ander op gerst gespecialiseerd haustoriumvormend pathogeen, nl. de dwergroestschimmel Puccinia hordei. Hoofdstuk 1 introduceert het onderwerp, basisresistentie van gerst tegen meeldauw, en geeft een korte beschrijving van de argumenten die suggereren dat basisresistentie, mlo-resistentie en niet-waard resistentie op moleculair niveau geassocieerd zijn. In Hoofdstuk 2 beschrijven we een QTL analyse die we uitvoerden om de genetische loci te vinden die bijdragen aan variatie in basisresistentie van gerst tegen meeldauw in het zaailingstadium en in het volwassenplantstadium. We deden deze analyse in zes karteringspopulaties van gerst. Die studie leidde tot de conclusie dat deze kwantitatieve resistentie gebaseerd is op een groot aantal genen. Slechts enkele daarvan werden gevonden in meer dan een populatie. De meeste van deze QTLs voor meeldauwresistentie zijn plantontwikkelingsstadium-specifiek. Uit genexpressie-, genexpressie onderdrukkings- en transformatie-experimenten kwamen 39 genen naar voren die bijdroegen aan de kans dat een meeldauwschimmel met succes een haustorium in een plantencel vormde. Deze genen kunnen worden beschouwd als kandidaatgenen die verantwoordelijk zijn voor natuurlijke verschillen in niveau van basisresistentie. We zochten sequentieverschillen in deze kandidaatgenen, zodat we hun kaartpositie konden bepalen. Dit lukte voor 23 van deze kandidaatgenen. Kaartposities van drie andere bij meeldauwresistentie betrokken genen, mlo , Ror1 en Ror2, waren al

119 Samenvatting

bekend, en ook deze werden voor onze studie beschouwd als kandidaatgenen voor basisresistentie. Vervolgens vergeleken we de kaartpositie van deze 26 kandidaatgenen met die van de gevonden QTLs voor basisresistentie. Daarvoor ontwikkelden we een verbeterde en zeer dichte, geïntegreerde koppelingskaart van gerst. Deze verbeterde kaart was gebaseerd op zeven karteringspopulaties van gerst. Meer dan 43% van de merkers die op deze kaart zijn samengebracht vertegenwoordigen gensequenties, en zijn van nut om in de toekomst kandidaatgenen te identificeren. Zes van de 26 kandidaatgenen hadden een kaartpositie die samenviel met die van een QTL voor basisresistentie tegen meeldauw. Deze zes kandidaatgenen zijn interessant voor vervolgstudies, omdat variatie in deze genen wel eens verantwoordelijk zouden kunnen zijn voor een deel van de verschillen in basisresistentie die gevonden werden in de karteringsstudies. Hoofdstuk 3 beschrijft de ontwikkeling van twee experimentele gerstlijnen met een extreem hoge en een extreem lage basisresistentie tegen gerstmeeldauw. Deze lijnen werden verkregen door toepassing van convergente kruisingen tussen respectievelijk de meest resistentie en tussen de meest vatbare lijnen in de karteringspopulaties die beschreven zijn in Hoofdstuk 2. We beschouwen de extreem vatbare en extreem resistente lijnen als waardevol materiaal voor verder onderzoek naar de moleculaire basis van basisresistentie tegen meeldauw. De resultaten wijzen erop dat fenotypische selectie voldoende effectief is om zeer hoge niveaus van basisresistentie te verkrijgen. We geven enkele argumenten dat merkergestuurde selectie wel eens onnodig en minder efficiënt zou kunnen zijn om een dergelijk hoog niveau van resistentie te bereiken. In Hoofdstuk 4 beschrijven we de ontwikkeling van twee andere experimentele gerstlijnen. Deze lijnen hebben, in het zaailingstadium, een niet eerder gemeld hoog niveau van vatbaarheid voor de op tarwe gespecialiseerde tarwemeeldauwschimmel. In een grote collectie gerstlijnen werd gezocht naar zeldzame gerstlijnen met een rudimentaire vatbaarheid voor dit tarwepathogeen. Die gerstlijnen werden in twee cycli onderling gekruist, wat resulteerde in twee buitengewone onderzoekslijnen, SusBgt SC en SusBgt DC . De kwantitatieve variatie tussen gerstlijnen en binnen de hier ontwikkelde nakomelingschappen wijst op een kwantitatieve overerving van deze niet-waard resistentie. Componentenanalyse van het infectieproces suggereerde dat factoren die de niet-waard resistentie van gerst tegen meeldauwvormen bepalen Blumeria - vorm-specifiek zijn. De ontwikkelde lijnen zullen dienen om de overerving van niet- waard resistentie in gerst tegen de tarwemeeldauw te bepalen. De lijnen zijn voorts een nuttig instrument in genexpressie- en complementatiestudies naar niet-waard resistentie. In Hoofdstuk 5 vermelden we dat een compatibele interactie tussen

120 Samenvatting

gerst en Puccinia hordei (de veroorzaker van dwergroest) de basisresistentie tegen een daarna aangebrachte meeldauw verhoogt. Deze pathogenen verschillen in manier waarop ze de afweer van plantencellen onderdrukken. We toonden aan dat het niveau van deze roest-geïnduceerde resistentie verschilt tussen gerstlijnen, en niet afhangt van het virulentiespectrum van het gebruikte meeldauwisolaat. Een macroarray genexpressie-experiment liet zien dat in de dubbel-geïnoculeerde behandeling verscheidene genen die een rol spelen in plant metabolisme en fotosynthese, verminderd tot expressie kwamen. Dit, terwijl enkele genen die een rol spelen in de afweer van planten versterkt tot expressie kwamen. Onder de laatstgenoemde zijn pathogenese gerelateerde eiwitten (PR-1) en cysteine synthase het vermelden waard. Onze histologische en genexpressiebevindingen zouden erop kunnen wijzen dat de eerdere infectie door de roest tot gevolg heeft dat genen voor basisresistentie tegen meeldauw worden “geprimed”, d.w.z. klaargemaakt om snel en sterk te reageren zodra een meeldauwschimmel de cel aanvalt. In Hoofdstuk 6 bespreken we de in de voorgaande hoofdstukken behaalde resultaten. Met name wijzen we op de perspectieven die de verkregen experimentele lijnen bieden voor de identificatie van genen die verantwoordelijk zijn voor basisresistentie van gerst tegen aangepaste en niet-aangepaste vormen van Blumeria graminis .

121 New perspectives to identify genes that…

122 Acknowledgments

Acknowledgments In the name of God, most gracious, most merciful. Looking back, I feel so fortunate of having had the opportunity to do my PhD at the Laboratory of Plant Breeding of Wageningen University. I did my PhD at a very friendly atmosphere and received continuous technical and emotional support during the last four years. It is a pleasure to convey my gratitude to all people who helped me in achieving this momentous task. First of all, I would like to express my deep sense of gratitude to my supervisor Dr. Rients Niks, for his patient guidance, encouragement and excellent advice throughout this study. Dear Rients, you were always ready to help. Your energetic personality and your enthusiasm for seeing new results have been stimulating me to improve my scientific background and also to enjoy my research outputs. You were so quick in commenting and correcting my written materials and your positive words gave me new confidence in myself and my ability. I will never forget your efforts to find a good living place for my family before our arrival and how happily you informed me that you could find a beautiful family house, a case of too-nice-to-be-true! You and your respectful wife, Marlien was very kind and supportive to me and my family. Thank you for all your help and support. I acknowledge my gratitude to Prof. Richard Visser for his valuable advices throughout my study. Dear Richard, thank you for stimulating discussions and for your comments and suggestions for the editing of my thesis. My sincere thanks to Thierry Marcel for his fruitful collaboration and his kind assistance in the research as well as for his valuable friendship. Dear Thierry, I have really enjoyed working in the same group with you and have learned a lot from you. I count you and your respectful wife, Hoa, among my best family friends. Many thanks go to Hossein Jafary and his respectful wife Shirin. Dear Hossein, I and my family will never forget your kind help and your warm and generous hospitality during the first days of our stay in the Netherlands. You kindly helped me to find my way in the Lab and easily shared your experience. Thank you for your support, help and friendship. My cordial thanks to Anton Vels for his kind assistance in setting up the field and the greenhouse trials. Dear Anton, I was so pleased to hear that you are recovering from your illness. You are indeed a great colleague. I am thankful to Annie Marchal, Letty Dijker and Mariame Gada at the secretary of Plant Breeding for dealing with administration and being always kind and helpful. I had the great fortune of sharing ideas with every member of resistance group, but more important, I have been honored with their friendship. It is a pleasure to mention

123 Acknowledgments

Sjaak van Heusden, Ben Vosman, Yuling Bai, Marieke Jeuken, Ningwen Zhang, Stefano Pavan, Luigi Faino, Zheng Zheng, Erik den Boer and Freddy Yeo. Furthermore, I would like to give special thanks to our Lab technicians, Petra vanden Berg, Fien Meijer-Dekens, Koen Pelgrom and Martijn van Kaauwen for creating such a nice atmosphere in the Lab. I am thankful to my friendly officemates Paul Dijkhuis, Awang Maharijaya, Firdaus Syarifin and Heungryul Lee. Extra thanks go to Awang for his kind help to design the cover of my thesis. I am grateful to Nadeem Khan, Arnold Kuzniar, Anitha Kumari, Sameer Joshi, Estelle Verzaux, Anoma Lokossou, Xingfeng Huang, Miqia Wang and Shital Dixit for their friendship. Dear Shital thank you also for your kind acceptance to be one of my paranimphs. My special thanks also go to Dr. Patrick Schweizer from IPK, Germany for his cooperation during the last two years and his great hospitality during my stay at IPK. I had a great opportunity to meet and establish friendship with many Iranian scientists in Wageningen. It is a pleasure to mention Dr. Mahmoud Otreshy, Rahim Mehrabi, Ramin Roohparvar, Mostafa Karbasioun, Benyamin Houshyani, Farhad Nazarian and their families. Dear Rahim and Farhad we shared various thoughts during our tea breaks and sport activities in the late afternoon together with Hossein. Thank you for your friendship. Furthermore I am grateful to Mahdi Arzanlou, Ali Haghnazari, Reza Talebi, Sara Dezhsetan, Reza Tadayonirad, Leila Samiei, Nasim Mansouri, Kambiz Baghalian and Alireza Babaei for their friendship. Dear Alireza, we shared many funny moments during our trips together, I value our friendship very much. It is a pleasure to convey my gratitude to Alireza Seifi. Dear Alireza, thank you for your encouragement and your kind help in the Lab as well as for creating such a great friendship. My special thanks go to Hedayat Bagheri, Mohammad Balali, Afshin Mehraban, Mahmoud Tabib Ghaffary and their families. Dear Hedayat and Mohammad, we have many good memories from our family trips and gatherings. I have found both of you trustworthy and honest friends. Dear Afshin and Mahmoud thank you for being always supportive to me and my family. You are very good friends. Furthermore, I want to thank Mr. and Mrs. Raei for their support and their kindness to me and my family. I would like to take this opportunity to thank my colleagues at Agricultural Research and Education Organization (AREO) and the Ministry of Science Research and Technology (MSRT) of my home country, IRAN, for financial support and also dealing

124 Acknowledgments

with a lot of official tasks during the last four years. Many thanks go in particular to the Staff Training Office of AREO and the scientific counselor of MSRT in Paris. I would like to express my special thanks to my brother-in-law Reza Tajy and my sister-in-law Maryam for their support and kindness. My PhD study could never be fulfilled without the full support from my family. I would like to express my profound gratitude to my beloved parents for their continuous emotional support and prayers during my study abroad. You made a long journey to visit us, indeed those were our best days in the Netherlands. To my lovely daughter, Mahrokh and my lovely son, Pouria, I feel the joy and happiness as watch you grow everyday. I am really proud of you! Finally I would like to express my deepest sense of gratitude to my wife, Fatemeh for her moral support and patience during my study. Dear Fatemeh, thank you for your generous love and persistent confidence in me. I love you so much.

Reza Aghnoum Wageningen, June 2009

125 Acknowledgments

123 About the author

About the author Reza Aghnoum was born on June 22, 1972 in Tehran, Iran but he grew up in a small city in the north east of Iran, where his parents had originally come from. After finishing his high- school, Reza started his academic studies in Plant Protection at Ferdowsi University of Mashhad in 1991. He followed a Master degree at the same university in Plant Pathology in 1995. Upon earning his Master degree in 1997, he was employed by the Agricultural Research and Education Organization (AREO) as a cereal pathologist-breeder at Mashhad Agricultural Research Center, where he worked for 7 years with a group of cereal breeders aiming to breed high yielding and disease resistant wheat and barley cultivars. In 2001 he passed the national examination and received a scholarship to do a PhD abroad. In February 2005 he started his PhD program at the Laboratory of Plant Breeding of Wageningen University under the supervision of Dr. Rients E. Niks and worked on basal resistance of barley to powdery mildew as his PhD thesis. Reza will join the scientific community of his home country, Iran, after finishing his PhD studies in the Netherlands.

Reza Aghnoum Khosaran Agricultural and Natural Resources Research Center P.O.Box: 91735-488 Mashhad, IRAN Email: [email protected] [email protected]

127 Acknowledgments

128 List of publications

List of publications

Marcel TC, Aghnoum R, Durand J, Varshney RK, Niks RE. 2007. Dissection of the barley 2L1.0 region carrying the ‘Laevigatum’ quantitative resistance gene to leaf rust using near isogenic lines (NIL) and subNIL. Molecular Plant–Microbe Interactions 20: 1604-1615.

Aghnoum R , Marcel TC, Johrde A, Pecchioni N, Schweizer P, Niks RE. Which candidate genes are responsible for natural variation in basal resistance of barley to barley powdery mildew? Submitted.

Aghnoum R , Niks RE. New perspectives to identify genes that determine non-host resistance of barley to non-adapted Blumeria graminis forms. Submitted.

Abstracts Aghnoum R, Marcel TC, Jafary H, Niks RE. Genetic diversity for quantitative resistance of barley against powdery mildew. Plant & Animal Genomes XV Conference. San Diego, California, USA. January 13-17, 2007.

Aghnoum R, Niks RE. Development of barley lines with extreme low and high level of basal resistance to powdery mildew.The 10 th International Barley Genetics Symposium. Alexandria, Egypt, April 5-10, 2008.

129 Acknowledgments

130 Education certificate

Education certificate

Education Statement of the Graduate School Experimental Plant Sciences

Issued to: Reza Aghnoum Date: 16 June 2009 Group: Plant Breeding, Wageningen University and Research Centre 1) Start-up phase date ► First presentation of your project Genetics of non-hypersensitive resistance of barley to powdery mildew Nov 1, 2005 ► Writing,or rewriting a project proposal Polygenic, non-hypersensitive resistance of barley to barley powdery mildew Feb-Aug 2005 ► Writing a review or book chapter ► MSc courses Genetic Analysis, Tools and Concepts (GEN-30306) Nov-Dec 2005 Breeding for Resistance (PBR 30306) Mar-Apr 2005 ► Laboratory use of isotopes Subtotal Start-up Phase 13.5 credits* 2) Scientific Exposure date ► EPS PhD student days EPS PhD student day 2005 in Radboud University Nijmegen Jun 2, 2005 SDV PhD student day 2006 in Paris Jun 9, 2006 EPS PhD student day 2006 in wageningen Sep 19, 2006 EPS PhD student day 2007 in wageningen Sep 13, 2007 1st Joint Retreat of the PhD Students Oct 2-3, 2008 in EPS ► EPS theme symposia EPS Theme 2 'Interactions between Plants and Biotic Agents, Utrecht University Jun 23, 2005 ► NWO Lunteren days and other National Platforms EPS - ALW Lunteren Apr 3-4, 2006 EPS - Lunteren meeting Apr 6-7, 2009 ► Seminars (series), workshops and symposia Molecular markers in yellow rust and powdery mildew: Dr. Xia-Yu Duan Nov 21, 2007 Plant Breeding Research day 2008, wageningen university, Jun 17, 2008 DNA demethylation in Arabidopsis:Prof.Jian-Kang Zhu Nov 3, 2008 Seminar: Prof. Nürnberger:Receptors in plant immunity Dec 17, 2008 ► Seminar plus ► International symposia and congresses Plant and Animal Genome XV Conference,USA Jan 13-17, 2007 Bioexploit meeting, Wageningen Mar 31-Apr 1, 2009 ► Presentations Poster: Plant and Animal Genome XV Conference,USA Jan 13-17, 2007 Poster:Plant Breeding Research day 2007, wageningen university, Sep 27, 2007 Poster:The 10th International Barley Genetics Symposium, Alexandria, Egypt Apr 5-10, 2008 Poster:Work shop "Natural Variation in plants", Aug 26-29, 2008 Oral presentation:Summerschool "On the Evolution of Plant Pathogen Interactions Jun 20, 2008 Poster:1st Joint Retreat of the PhD Students in EPS Oct 2-3, 2008 Oral presentation:EPS-Lunteren meeting Apr 7, 2009

► IAB interview Sep 14, 2007 ► Excursions Excursion to Barenburg grass breeding company, Wolfheze, Oct 2,2007 Excursion to Bejo seeds and Royal van Zanten companies, Jun 11, 2008 Excursion to Syngenta company in Einkhuzen Sep 25, 2008 Subtotal Scientific Exposure 10.4 credits* 3) In-Depth Studies date ► EPS courses or other PhD courses Summerschool "Signalling in Plant Development and Plant Defense" Jun 21-23, 2006 Summerschool "On the Evolution of Plant Pathogen Interactions Jun 18-20, 2008 EPS PhD Work shop "Natural Variation in plants", Aug 26-29, 2008 ► Journal club Litterature discussion in Department of Plant Breeding 2005-2008 ► Individual research training Two weeks Lab training on gene expression analysis, IPK Germany Jan 25-Feb 06, 2009 Subtotal In-Depth Studies 9.0 credits* 4) Personal development date ► Skill training courses Working with EndNote 8, Wageningen UR Library Mar 8, 2005 Techniques for writing and presenting a scientific paper Oct 16-19, 2007 ► Organisation of PhD students day, course or conference ► Membership of Board, Committee or PhD council Subtotal Personal Development 1.4 credits* TOTAL NUMBER OF CREDIT POINTS* 34.3 Herewith the Graduate School declares that the PhD candidate has complied with the educational requirements set by the Educational Committee of EPS which comprises of a minimum total of 30 credits * A credit represents a normative study load of 28 hours of study

131 Acknowledgments

This research has been carried out at the Laboratory of Plant Breeding of Wageningen University and finantially was supported by the Agricultural Research and Education Organization (AREO) and the Ministry of Science Research and Technology of I. R. of Iran.

Thesis layout: by the author Cover design: by Awang Maharijaya and the author Front page: schematically represents the complexity of the papilla-based defense Back page: abundant conidiation by the non-adapted wheat powdery mildew on the barley line SusBgt SC

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