Genome-Wide Study of the Tomato SlMLO Gene Family and Its Functional Characterization in Response to the Powdery Mildew Fungus Oidium neolycopersici

Abstract

The MLO (Mildew Locus O) gene family encodes plant-specific proteins containing seven transmembrane domains and likely acting in signal transduction in a calcium and calmodulin dependent manner. Some members of the MLO family are susceptibility factors toward fungi causing the powdery mildew disease. In tomato, for example, the loss-of-function of the MLO gene SlMLO1 leads to a particular form of powdery mildew resistance, called ol-2, which arrests almost completely fungal penetration. This type of penetration resistance is characterized by the apposition of papillae at the sites of plant-pathogen interaction. Other MLO homologs in Arabidopsis regulate root response to mechanical stimuli (AtMLO4 and AtMLO11) and pollen tube reception by the female gametophyte (AtMLO7). However, the role of most MLO genes remains unknown. In this work, we provide a genome-wide study of the tomato SlMLO gene family. Besides SlMLO1, other 15 SlMLO homologs were identified and characterized with respect to their structure, genomic organization, phylogenetic relationship, and expression profile. In addition, by analysis of transgenic plants, we demonstrated that simultaneous silencing of SlMLO1 and two of its closely related homologs, SlMLO5 and SlMLO8, confer higher level of resistance than the one associated with the ol-2 mutation. The outcome of this study provides evidence for functional redundancy among tomato homolog genes involved in powdery mildew susceptibility. Moreover, we developed a series of transgenic lines silenced for individual SlMLO homologs, which lay the foundation for further investigations aimed at assigning new biological functions to the MLO gene family.

Keywords: MLO gene family; powdery mildew disease; susceptibility; tomato.

Figure 1

Rooted circular cladogram showing the phylogenetic relationships of the tomato SlMLO proteins. A UPGMA-based tree comprises all the sequences of the MLO protein family of Arabidopsis (At) and tomato (Sl). Individual sequences of apple (Md), tobacco (Nt), cucumber (Csa), pea (Ps), Lotus japonicus (Lj), barrel clover (Mt), pepper (Ca), barley (Hv), rice (Os), and wheat (Ta) are included. Numbers on each node represent bootstrap values based on 100 replicates. Phylogenetic clades are designated with colors and Roman numbers according to the position of AtMLO homologs and apple MdMLO, as indicated by Pessina et al. (2014). The tomato SlMLO protein sequences used for this tree derived all from the translation of the transcripts cloned from leaf of the cv. Moneymaker, except for SlMLO12, which corresponds to the translated sequence of flower. Accession numbers of the sequences used, other than tomato SlMLO, are listed in Supplementary Table 2.

Figure 2

Relative expression level of SlMLO transcripts evaluated in four different tissues (leaf, root, flower, and mature fruit) of the tomato cv. Moneymaker in axenic condition. The expression level of each gene is compared to the abundance of Ef1α which was used as reference gene. Bars show standard errors based on three technical replicates. Similar trends are reported in Supplementary Figure 3.

Figure 3

Relative expression level of the SlMLO gene family in response to O. neolycopersici inoculation. Samples were collected at 0, 6, and 10 h after inoculation (hpi). Transcript abundance of each SlMLO homolog was normalized against the transcription level of the 60S ribosomal protein L33 used as reference gene. Bars show standard errors based on four biological replicates. Asterisks refer to significant differences with respect to non-inoculated plants (0 hpi), inferred by mean comparisons with a Student's t-test (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). The SlMLO genes harbored in clade V, based on the phylogenetic tree of Figure 1, are indicated by an arrow spanning their corresponding bars. Similar results were obtained by using the elongation factor Ef1α as housekeeping gene (Supplementary Figure 4).

Figure 4

Evaluation of the silencing effect of the RNAi constructs designed to target SlMLO1, SlMLO3, SlMLO5, and SlMLO8 in segregating T₂ families of the tomato cv. Moneymaker. Panels (A–D) show the expressions of clade V SlMLO homologs in plants of T₂ families, derived from different transformation events and segregating for the presence [T₂_NPT(+)] or absence [T₂_NPT(-)] of the RNAi::SlMLO1, RNAi::SlMLO3, RNAi::SLMLO5, and RNAi::SlMLO8 constructs, respectively. In (A) bars and standard errors refer to eight plants T₂_NPT(+) and four plants T₂_NPT(-) of two T₂ families and four Moneymaker (MM) plants. In (B) bars and standard errors refer to ten plants T₂_NPT(+) and five plants T₂_NPT(-) of three T₂ families and four MM individuals. In (C) bars and standard errors refer to ten plants T₂_NPT(+) and five plants T₂_NPT(-) of three T₂ families and four MM individuals. In (D) bars and standard errors refer to 10 T₂_NPT(+) of two T₂ families and four MM individuals.

Figure 5

Powdery mildew evaluation on plants of segregating T₂ families obtained with silencing constructs targeting SlMLO genes to attest their involvement in O. neolycopersici susceptibility. Panel (A) shows the phenotypic evaluation of the powdery mildew growth on leaves of different T₂ individuals that have been evaluated for the (from left to right) absence of the RNAi::SlMLO1, presence of the RNAi::SlMLO1, presence of the RNAi::SlMLO3, presence of the RNAi::SlMLO5, and presence of the RNAi::SlMLO8 silencing constructs, followed by one individual of the cv Moneymaker (MM) and one of the Slmlo1 line carrying a loss-of-function mutation in the SlMLO1 gene. Panel (B) shows the relative quantification of the ratio between Oidium neolycopersici and plant gDNAs in transgenic individuals [NPT(+)] and not transgenic individuals [NPT(-)] segregating in T₂ families obtained with the silencing constructs above described. Bars and standard errors refer to (from left to right) four individuals of two independent T₂ families not carrying the RNAi::SlMLO1, eight individuals of the same two T₂ families carrying the RNAi::SlMLO1, 18 individuals of three independent T₂ segregating families carrying the RNAi::SlMLO3 construct, 18 individuals of three independent T₂ segregating families carrying the RNAi::SlMLO5 construct and 20 individuals of two T₂ segregating families carrying the RNAi::SlMLO8 construct, next to 10 MM plants and 10 plants of the Slmlo1 line. The asterisk refers to the significant difference in susceptibility between individuals of the T₂_SlMLO1_NPT(+) and Slmlo1, inferred by mean comparisons with a Student's t-test (^*p < 0.05).

Figure 6

Effect of the silencing of SlMLO1, SlMLO5, and SlMLO8 in tomato cv Moneymaker background compared with the Slmlo1 line harboring a loss-of-function of the SlMLO1 gene. Panel (A) refers to a transgenic plant carrying the RNAi::SlMLO1 construct, Panel (B) a plant of the Slmlo1 line and (C) a plant of the tomato cv. Moneymaker. Panels (A–C) show fungal structures (spores, germination tube, appressorium, haustorium –HS-, and hyphae) and the plant cellular reaction of papilla apposition at the sites of fungal penetration.