Exploring recessive resistance to the powdery mildew disease

The powdery mildew disease, caused by obligate biotrophic fungi belonging to the Ascomycete order of Erysiphales, is common among higher plants and represents one of the most important threats for the cultivation of many crop species. Although powdery mildew resistance is usually a dominant trait, recessively inherited resistance has been reported to occur in Arabidopsis, barley, tomato and pea. In Chapter 1 of this thesis, we provide a state of the art on the understanding of mechanisms underlying plant immunity and review monogenic dominant and recessive sources used in breeding in order to develop resistant cultivars. In Chapter 2 and 3, we describe a successful homology-based cloning approach for the isolation of the recessive ol-2 gene, naturally occurring in a tomato accession collected in Ecuador and conferring broad-spectrum resistance to the powdery mildew fungus Oidium neolycopersici. We realized that ol-2 resistance shares striking similarities with well-known barley and Arabidopsis mlo powdery mildew resistance, originating from loss-of-function mutations of genes encoding for specific seven transmembrane domains MLO isoforms. The following chain of evidence was provided demonstrating that ol-2 resistance is due to the loss of the tomato MLO homolog SlMLO1: a) Ol-2 and SlMLO1 loci share the same genetic and cytogenetic position; b) resistant ol-2/ol-2 lines are homozygous for a loss-of-function deletion in the sequence of SlMLO1; c) a molecular marker developed on the mutation site co-segregates with resistant individuals in an F2population; d) SlMLO1 transgenic expression in ol-2/ol-2 individuals results in disease susceptibility; e) SlMLO1 virus-induced silencing in Ol-2/Ol-2 individuals is associated to increased powdery mildew resistance. In Chapter 4, we first illustrate a chemical mutagenesis program allowing the identification of a pea line showing recessive resistance towards the powdery mildew fungus Erysiphe pisi. Histological and genetic analyses revealed that the mutated gene is allelic to er1, commonly used in pea breeding for the development of resistant cultivars. As defense mechanisms associated to er1 resistance are reminiscent of mlo immunity, we sequenced the pea MLO homolog PsMLO1 and found a loss-of-function point mutation characterizing the resistant line. A polymorphic CAPS marker was developed on the mutation site and found to be fully co-segregating with resistance in a large F2population. Finally, PsMLO1 sequencing in three er1 resistant cultivars also resulted in the identification of aberrant alleles, further substantiating the identification of another case of mlo-based immunity. In Chapter 5, we report the identification (in vitro and in silico) of a series of MLO homolog sequences in five cultivated Solanaceae species affected by the powdery mildew disease. Comparative analyses using a dataset of several dicot MLO proteins allowed the identification of candidate isoforms for disease susceptibility and the detection of cluster-specific transmembrane amino acid motifs. In Chapter 6, we look at disease resistance as a condition due to the lack of susceptibility genes like MLO. We review several susceptibility genes isolated in crop species and in Arabidopsis, with respect to their molecular characterization, their role in plant-pathogen interactions and the resistant phenotype deriving from their loss-of-function mutations/silencing. A breeding strategy based on the lack of plant susceptibility genes is discussed.