Research

Molecular basis of race specific resistance in cereals

In cereals, race-specific resistance genes, including those effective against powdery mildew diseases, are an agronomically important resource for breeding. Race-specific resistance is conditioned by the interaction between a resistance (R) gene from the host (typically encoding for an NBS-LRR receptor), and an avirulence (Avr) gene from the pathogen (typically encoding for an effector). We are interested in understanding the genetic, molecular, and biochemical bases of such resistance in cereals. More specifically, we focus on the interaction between powdery mildew resistance proteins (PM) from wheat and avirulence effector proteins (AVRPM) from the pathogenic fungus Blumeria graminis.

 

(A) Suppression of the AvrPm3a2/f2-Pm3f mediated hypersensitive cell death response (revealed by Trypan blues staining) in presence of the SvrPm3a1/f1 suppressor; (B) 3D computational modelling reveals a horse-shoe structure of the leucine-rich-repeats (LRR) region of the PM3 resistance protein; (C) Suppression of the hypersensitive cell death response mediated by the auto-activated Pm8 resistance gene in presence of the susceptible allele of the Pm3 resistance gene (Pm3CS).

(A) Suppression of the AvrPm3a2/f2-Pm3f mediated hypersensitive cell death response (revealed by Trypan blues staining) in presence of the SvrPm3a1/f1 suppressor; (B) 3D computational modelling reveals a horse-shoe structure of the leucine-rich-repeats (LRR) region of the PM3 resistance protein; (C) Suppression of the hypersensitive cell death response mediated by the auto-activated Pm8 resistance gene in presence of the susceptible allele of the Pm3 resistance gene (Pm3CS).

 

Our lab has been involved in the cloning and characterization of several Pm genes including Pm2, Pm8, and seventeen alleles of the Pm3 gene. We have also cloned the first mildew avirulence effector AvrPm3a2/f2, as well as the first suppressor of race-specific resistance SvrPm3a1/f1. We employ next-generation sequencing (NGS) and high-throughput genotyping (HTG) technologies to genetically map the loci encoding for mildew resistance genes in cereals, and avirulence/suppressor genes in powdery mildews. We use Nicotiana benthamiana, Arabidopsis thaliana, Agrobacterium tumefaciens, and Escherichia coli as heterologous systems to deploy a variety of molecular/biochemical assays to study the basis of race specific resistance down to the single amino-acid level.

 

Our current research aims at understanding the molecular and biochemical basis of AVR-R recognition and downstream resistance signaling. In another line of research, we want to understand the mechanisms leading to resistance suppression as a result of molecular interaction between PM proteins, or the action of pathogen encoded suppressors.

 

[1] Bourras et al. (2015) Multiple avirulence loci and allele-specific effector recognition control the Pm3 race-specific resistance of wheat to powdery mildew. The Plant Cell 27: 2991-3012. Link

[2] Hurni et al. (2014) The powdery mildew resistance gene Pm8 derived from rye is suppressed by its wheat ortholog Pm3. The Plant Journal 79: 904-913. Link

[3] Stirnweis at al. (2014) Suppression among alleles encoding nucleotide-binding–leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants. The Plant Journal 79: 893-903. Link

 

 

Molecular basis of broad spectrum quantitative resistance in cereals

We want to understand the molecular basis underlying the mode of action of broad-spectrum quantitative resistance genes in cereals. The agronomical importance of such resistance genes is illustrated by Lr34, a gene that has been effective over the past 50 years in conferring quantitative durable resistance to fungal pathogens in wheat. Lr34, which encodes for an ABC-transporter protein, has been cloned in our lab and is a major focus of our research. We showed that this gene is effective not only in wheat, but also in other cereals such as barley and rice. We are specifically interested in the biochemical processes regulated by this ABC-transporter, and their role in pathogen resistance.

 

In addition to Lr34, we have been involved in the cloning of Htn1, a resistance gene of maize against northern corn leaf blight. In another line of research, we are also investigating the molecular basis of the Lr75-mediated resistance of wheat to leaf rust. We combine genetic crosses and EMS mutagenesis with high-throughput genotyping, NGS technologies, and QTL mapping to identify novel broad-spectrum resistance genes. These approaches are complemented by field studies and the production of stable transgenic plants. In addition, we rely on heterologous assays in N. benthamiana, A. thaliana, and yeast, to study the biochemical functions/pathways underlying the resistance mechanism.

 

Our current research aims at unraveling the major genetic, molecular, and biochemical components of broad spectrum resistance in cereals, based on the Lr34, Lr75, and Htn1 systems we are studying.

 

[1] Krattinger et al. (2015) The wheat durable, multipathogen resistance gene Lr34 confers partial blast resistance in rice. Plant Biotechnology journal 14: 1261-1268. Link

[2] Hurni et al. (2015) The maize disease resistance gene Htn1 against northern corn leaf blight encodes a wall-associated receptor-like kinase. PNAS 112: 8780-8785. Link

[3] Chauhan et al. (2015) The wheat resistance gene Lr34 results in the constitutive induction of multiple defense pathways in transgenic barley. The Plant Journal 84:202-215. Link

 

 

Pathogenomics of cereal mildews

The study of pathogenomics in cereal mildews attempts to utilize genome-wide information generated by NGS technologies to understand the genetic and molecular basis of disease. One major goal of our studies is to assess the genomic and transcriptomic diversity of mildew isolates from across the world.

 

We have produced the reference genome of wheat powdery mildew that is a pre-requisite for applying large-scale NGS approaches. We have sequenced and analyzed over 20 isolates from different geographical origins, and we are regularly acquiring new mildew accessions from a variety of hosts, and from around the world for additional sequencing. We found that many gene families commonly involved in primary and secondary metabolism in fungi were absent from powdery mildews. We have also found that mildew diversity is relatively small and that mildew populations are mosaics of ancient haplotypes that have evolved long before the onset of agriculture. Our current research aims at expanding these analyses to other formae speciales of cereal mildews to better understand the mechanisms shaping genetic diversity.

 

In another line of research, we are interested in understanding mildew virulence at the gene expression level. We have applied three isolates of mildew to RNA sequencing and small RNA sequencing, and acquired data from infected and non-infected wheat material. There, our focus is to study whole transcriptome diversity, in particular among the effector complements of different isolates. On the molecular level, we are also using RNA interference approaches to target highly expressed or essential virulence gene in mildews.

 

[1] Wicker et al. (2013) The wheat powdery mildew genome shows the unique evolution of an obligate biotroph. Nature Genetics 45: 1092-1096. Link

[2] Parlange et al. (2011) A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome. Functional & Integrative Genomics 11: 671–677. Link

 

 

Host-adaptation and evolution of cereal mildews

Blumeria graminis (B.g.), commonly known as grass powdery mildew, is a fungal pathogen that attacks cultivated cereals as well as wild grasses. There are several formae speciales (literally special forms) each of which is adapted to a single host such as B.g. f.sp. tritici on wheat, B.g. f.sp. hordei on barley, and B.g. f.sp. secalis on rye. We are interested in studying the emergence and evolution of mildew formae speciales, and identifying the molecular basis of host specialization in this major class of plant pathogens. In particular, we are interested in identifying genes that are responsible for host specificity, host range expansions, and host jumps.

 

Recently, a new forma specialis has emerged on triticale which is an artificial hybrid between wheat and rye that remained immune to grass mildews until 2000. One of our major finding was that B.g. f.sp. triticale is actually a hybrid between wheat and rye powdery mildews, same as the triticale host is a hybrid between wheat and rye. We are now investigating the genetic basis of this hybrid gain of virulence using genetic crosses, NGS, and QTL mapping approaches.

 

In another line of research, we are investigating the genetic basis of powdery mildew virulence towards immune-deficient mutants of the non-host Arabidopsis thaliana. These mutants allow successful host penetration and micro-colony formation for some B.g. tritici isolates, thus providing a genetic system for studying non-host resistance of this dicot host to grass mildews.

 

[1] Menardo et al. (2016) Hybridization of powdery mildew strains gives rise to pathogens on novel agricultural crop species. Nature Genetics 48: 201-205. Link

[2] Wicker et al. (2013) The wheat powdery mildew genome shows the unique evolution of an obligate biotroph. Nature Genetics 45: 1092-1096. Link

[3] Oberhaensli et al. (2011) Comparative sequence analysis of wheat and barley powdery mildew fungi reveals gene colinearity, dates divergence and indicates host-pathogen co-evolution. Fungal Genetics and Biology 48:327-334. Link

 

 

Cereal genomics and genetic diversity

Comparative genomics focuses on the large-scale comparison of entire genomes. Typically, we compare gene content and gene order between two or more organisms. In cereals, comparative genomics is a powerful method to discover mechanisms that drive the evolution of these extremely complex genome. We have discovered multiple mechanisms that can lead to gene duplications, gene movement and gene loss. One central mechanism driving genome rearrangements is DNA double-strand breaks (DSB) which can be introduced by transposable elements (TE) activity. In plants, DSB repair is often error-prone, leading to the deletion, translocation and duplication of large genomic segments. Thus, one major focus of our research is to investigate the role of transposable elements activity in the evolution of cereal genomes where TEs represent up to 90% of the genome sequence.

Picture for Cereal genomics and genetic diversity

 

In another line of research, we are interested in studying the genetic diversity of cereals. We are currently assessing a large collection of wheat, spelt wheat, and barley accessions and searching for genetic variations that could be linked to agronomically important traits. To do so, we use several NGS-based methodologies for rapid and high-throughput genotyping of a large number of accessions. Together with phenotypical information (e.g., disease resistance), those data can be used to identify alleles and/or genes which are interesting for breeders.

 

[1] Middleton et al. (2014) Sequencing of chloroplast genomes from wheat, barley, rye and their relatives provides a detailed insight into the evolution of the Triticeae tribe. PLoS ONE doi: 10.1371/journal.pone.0085761. Link

[2] Buchmann et al. (2012) Interspecies sequence comparison in Brachypodium reveals how transposon activity corrodes genome colinearity. The Plant Journal 71: 550-563. Link

[3] Wicker et al. (2011) Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley and their relatives. The Plant Cell 23: 1706-1718. Link