Discovery and Characterization of a Novel Tomato mlo Mutant from an EMS Mutagenized Micro-Tom Population

Abstract

In tomato (Solanum lycopersicum), there are at least three SlMLO (Mildew resistance Locus O) genes acting as susceptibility genes for the powdery mildew disease caused by Oidium neolycopersici, namely SlMLO1, SlMLO5 and SlMLO8. Of the three homologs, the SlMLO1 gene plays a major role since a natural mutant allele called ol-2 can almost completely prevent fungal penetration by formation of papillae. The ol-2 allele contains a 19-bp deletion in the coding sequence of the SlMLO1 gene, resulting in a premature stop codon within the second cytoplasmic loop of the predicted protein. In this study, we have developed a new genetic resource (M200) in the tomato cv. Micro-Tom genetic background by means of ethyl methane sulfonate (EMS) mutagenesis. The mutant M200 containing a novel allele (the m200 allele) of the tomato SlMLO1 gene showed profound resistance against powdery mildew with no fungal sporulation. Compared to the coding sequence of the SlMLO1 gene, the m200 allele carries a point mutation at T65A. The SNP results in a premature stop codon L22* located in the first transmembrane domain of the complete SlMLO1 protein. The length of the predicted protein is 21 amino acids, while the SlMLO1 full-length protein is 513 amino acids. A high-resolution melting (HRM) marker was developed to distinguish the mutated m200 allele from the SlMLO1 allele in backcross populations. The mutant allele conferred recessive resistance that was associated with papillae formation at fungal penetration sites of plant epidermal cells. A comprehensive list of known mlo mutations found in natural and artificial mutants is presented, which serves as a particularly valuable resource for powdery mildew resistance breeding.

Keywords: EMS mutagenesis; Micro-Tom; Oidium neolycopersici; SlMLO1; Solanum lycopersicum; powdery mildew.

Figure 1

Pedigree scheme of (A) F₄ plants homozygous for the m200 allele, (B) BC₃S₂ lines homozygous for the ol-2 allele in Solanum lycopersicum cv. Moneymaker background (ol-2_MM) and (C) F₄ plants carrying the ol-2 allele in cv. Super Marmande background (ol-2_SM).

Figure 2

A novel EMS mlo mutant (M200) shows resistance to powdery mildew. (A) Contrasting phenotypes of susceptible leaves of an M₁ plant and resistant leaves of the M200 plant after Oidium neolycopersici inoculation. (B) Schematic representation of the SlMLO1 protein of the cv. Heinz. The predicted truncated m200 protein is indicated in yellow, while the region that is absent in m200 is indicated in grey.

Figure 3

Phenotypic evaluation of the powdery mildew symptoms and relative fungal biomass quantification. Panel (A) shows leaves collected 18 days after the pathogen inoculation. Panel (B) refers to fungal biomass measured by relative quantification of the ratio between Oidium neolycopersici and plant gDNAs on different genotypes (F₄ plants carrying the m200 allele, plants carrying the ol-2 allele in Moneymaker (MM) and Super Marmande background, a plant carrying the RNAi::SlMLO1 construct, and MM). Bars show standard errors based on four plants. Columns labeled with different letters are significantly different at p < 0.05 according to Tukey’s multiple comparison test.

Figure 4

Microscopic observations on powdery mildew infection and development of the infection units (IU) of Oidium neolycopersici on six different genotypes. In each panel photos are taken from (A) and (B) F₄ plants carrying the m200 allele, (C) a plant carrying the RNAi::SlMLO1 construct, (D) and (E) plants carrying the ol-2 allele in Moneymaker (MM) and Super Marmande (SM), (F) MM, respectively. Photos of two IU/genotype are shown, except for the MM and F₄-2 carrying the m200 allele where only one IU is shown. Each photo is taken with different focus to observe all the fungal structures and papillae, from the most superficial to the deepest ones. Sp = spore, AP = appressorium; HS = haustorium; 2 hyphae = secondary hyphae.

Figure 5

Schematic representation of the complete barley HvMLO protein. The orange bar represents the plant membrane. Colored dots indicate the amino acids of the corresponding mlo-mutants in different plant species. Overview of the depicted mlo-mutants are shown in Table S4.