Map- vs. homology-based cloning for the recessive gene ol-2 conferring resistance to tomato powdery mildew

The recessive gene ol-2 confers papilla-associated and race-non-specific resistance to tomato powdery mildew caused by Oidium neolycopersici. In order to facilitate marker assisted selection (MAS) in practical breeding programmes, we identified two simple sequence repeat (SSR) markers and one cleaved amplified polymorphic sequence (CAPS) marker which are linked to the resistance locus and co-dominantly inherited. Aiming to provide a base for ol-2 positional cloning, we used a large segregating F2 population to merge these markers with all the ol-2 linked amplified fragment length polymorphism (AFLP®) markers previously identified in an integrated genetic map. By screening a tomato bacterial artificial chromosome (BAC) library, we detected two BAC clones containing two expressed sequence tags (ESTs) homologous to the gene mlo, responsible for powdery mildew resistance in barley, as well as an ol-2-linked marker. Chromosomal mapping by Fluorescence in situ Hybridization (FISH) revealed major signals of the two BAC DNAs in the pericentromeric heterochromatin of the short arm of chromosome 4, in the same region where the ol-2 gene was previously mapped. The genetic and cytogenetic co-localisation between ol-2 and tomato mlo-homologue(s), in addition to the similarity of ol-2 and mlo resistances for both genetic and phytopathological characteristics, suggests that ol-2 is likely a mlo-homologue. Thus, a homology-based cloning approach could be more suitable than positional cloning for ol-2 isolation

Loss of Function in Mlo Orthologs Reduces Susceptibility of Pepper and Tomato to Powdery Mildew Disease Caused by Leveillula taurica

Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse tomato and pepper production. In contrast to most powdery mildew species which are epiphytic, L. taurica is an endophytic fungus colonizing the mesophyll tissues of the leaf. In barley, Arabidopsis, tomato and pea, the correct functioning of specific homologues of the plant Mlo gene family has been found to be required for pathogenesis of epiphytic powdery mildew fungi. The aim of this study was to investigate the involvement of the Mlo genes in susceptibility to the endophytic fungus L. taurica. In tomato (Solanum lycopersicum), a loss-of-function mutation in the SlMlo1 gene results in resistance to powdery mildew disease caused by Oidium neolycopersici. When the tomato Slmlo1 mutant was inoculated with L. taurica in this study, it proved to be less susceptible compared to the control, S. lycopersicum cv. Moneymaker. Further, overexpression of SlMlo1 in the tomato Slmlo1 mutant enhanced susceptibility to L. taurica. In pepper, the CaMlo2 gene was isolated by applying a homology-based cloning approach. Compared to the previously identified CaMlo1 gene, the CaMlo2 gene is more similar to SlMlo1 as shown by phylogenetic analysis, and the expression of CaMlo2 is up-regulated at an earlier time point upon L. taurica infection. However, results of virus-induced gene silencing suggest that both CaMlo1 and CaMlo2 may be involved in the susceptibility of pepper to L. taurica. The fact that overexpression of CaMlo2 restored the susceptibility of the tomato Slmlo1 mutant to O. neolycopersici and increased its susceptibility to L. taurica confirmed the role of CaMlo2 acting as a susceptibility factor to different powdery mildews, though the role of CaMlo1 as a co-factor for susceptibility cannot be excluded

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. </p

Solanum lycopersicum (Tomato)

After its introduction in Europe the tomato (Solanum lycopersicum) has gone a long way. Dedicated breeding has resulted in numerous cultivars grown all over the world, differing in all kind of aspects such as yield, shape, resistance, taste and quality. Modern cultivars are sold as hybrids with a very good performance. Since some decades the genetic variation of tomato and wild tomato relatives is conserved and successfully exploited to introgress genes resulting in tomatoes better able to cope with biotic and abiotic stress. Tomato has become a model species for genetic and genomic studies and the sequence of the gene-rich regions will be determined in the near futur

Solanum lycopersicum (Tomato)

After its introduction in Europe the tomato (Solanum lycopersicum) has gone a long way. Dedicated breeding has resulted in numerous cultivars grown all over the world, differing in all kind of aspects such as yield, shape, resistance, taste and quality. Modern cultivars are sold as hybrids with a very good performance. Since some decades the genetic variation of tomato and wild tomato relatives is conserved and successfully exploited to introgress genes resulting in tomatoes better able to cope with biotic and abiotic stress. Tomato has become a model species for genetic and genomic studies and the sequence of the gene-rich regions will be determined in the near futur

Pea powdery mildew er1 resistance is associated to loss-of-function mutations at a MLO homologous locus

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F2 individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection

Identification of a complete set of functional markers for the selection of er1 powdery mildew resistance in Pisum sativum L.

Powdery mildew is the most widespread disease of pea (Pisum sativum L.) and causes severe economic losses worldwide. Recessively inherited er1 powdery mildew resistance, successfully used for decades in pea breeding programs, has recently been shown to originate from the loss of function of the PsMLO1 gene. Five er1 alleles, each corresponding to a different PsMLO1 null mutation, have been characterized to date in pea germplasm. In order to aid er1 selection, we aimed to identify functional markers which target PsMLO1 polymorphisms directly responsible for the resistant phenotype. Highly informative cleaved amplified polymorphic sequence (CAPS), derived cleaved amplified polymorphic sequence (dCAPS), sequence tagged site (STS) and highresolution melting (HRM) markers were developed which enable the selection of each of the five er1 alleles. Taken together, the results described here provide a powerful tool for breeders, overcoming limitations of previously reported er1-linked markers due to the occurrence of recombination with the resistance locus and/or the lack of polymorphism between parental genotypes. The HRM marker er1-5/HRM54 reported here, targeting a mutagenesisinduced er1 allele recently described by us, does not require manual processing after PCR amplification, and is therefore suitable for large-scale breeding programs based on high-throughput automated screening

Monocot and dicot MLO powdery mildew susceptibility factors are functionally conserved in spite of the evolution of class-specific molecular features

Background Specific members of the plant Mildew Locus O (MLO) protein family act as susceptibility factors towards powdery mildew (PM), a worldwide-spread fungal disease threatening many cultivated species. Previous studies indicated that monocot and dicot MLO susceptibility proteins are phylogenetically divergent. Methods A bioinformatic approach was followed to study the type of evolution of Angiosperm MLO susceptibility proteins. Transgenic complementation tests were performed for functional analysis. Results Our results show that monocot and dicot MLO susceptibility proteins evolved class-specific conservation patterns. Many of them appear to be the result of negative selection and thus are likely to provide an adaptive value. We also tested whether different molecular features between monocot and dicot MLO proteins are specifically required by PM fungal species to cause pathogenesis. To this aim, we transformed a tomato mutant impaired for the endogenous SlMLO1 gene, and therefore resistant to the tomato PM species Oidium neolycopersici, with heterologous MLO susceptibility genes from the monocot barley and the dicot pea. In both cases, we observed restoration of PM symptoms. Finally, through histological observations, we demonstrate that both monocot and dicot susceptibility alleles of the MLO genes predispose to penetration of a non-adapted PM fungal species in plant epidermal cells. Conclusions With this study, we provide insights on the evolution and function of MLO genes involved in the interaction with PM fungi. With respect to breeding research, we show that transgenic complementation assays involving phylogenetically distant plant species can be used for the characterization of novel MLO susceptibility genes. Moreover, we provide an overview of MLO protein molecular features predicted to play a major role in PM susceptibility. These represent ideal targets for future approaches of reverse genetics, addressed to the selection of loss-of-function resistant mutants in cultivated species