Michela Appiano, Stefano Pavan, Domenico Catalano, Zheng Zheng, Valentina Bracuto, Concetta Lotti, Richard G. F. Visser, Luigi Ricciardi, Yuling Bai

Trangenic Research, May 2015

Abstract

Specific homologs of the plant Mildew Locus O (MLO) gene family act as susceptibility factors towards the powdery mildew (PM) fungal disease, causing significant economic losses in agricultural settings. Thus, in order to obtain PM resistant phenotypes, a general breeding strategy has been proposed, based on the selective inactivation of MLO susceptibility genes across cultivated species. In this study, PCR-based methodologies were used in order to isolate MLO genes from cultivated solanaceous crops that are hosts for PM fungi, namely eggplant, potato and tobacco, which were named SmMLO1, StMLO1 and NtMLO1, respectively. Based on phylogenetic analysis and sequence alignment, these genes were predicted to be orthologs of tomato SlMLO1 and pepper CaMLO2, previously shown to be required for PM pathogenesis. Full-length sequence of the tobacco homolog NtMLO1 was used for a heterologous transgenic complementation assay, resulting in its characterization as a PM susceptibility gene. The same assay showed that a single nucleotide change in a mutated NtMLO1 allele leads to complete gene loss-of-function. Results here presented, also including a complete overview of the tobacco and potato MLO gene families, are valuable to study MLO gene evolution in Solanaceae and for molecular breeding approaches aimed at introducing PM resistance using strategies of reverse genetics.

Fig. 2 Effects of the transgenic expression ofNtMLO1in a tomatomloloss-of-function genetic background.aFrom left to right as follows: one individual of a T2 family positive for the presence the NtMLO1 overexpression construct [T2_a(?)]; one individual of another independent T2 family positive for the presence of the NtMLO1 overexpression construct [T2_b(?)]; one T2 individual negative for the presence of the overexpres-sion construct [T2(-)]; one individual of the tomato Slmlo1 mutant line, carrying a loss of function deletion in the SlMLO1 gene; one individual of the susceptible cultivar Moneymaker (MM). b Reports the average visual scoring of disease incidence observed on the individuals of the same two T2 families [T2_a(?) and T2_b(?)]; individuals of both T2_a and T2_b families negative for the presence of the 35S::NtMLO1 construct [T2(-)]; individuals of the Slmlo1 mutant line; individuals of the cultivar MM. The scale from 0 completely resistant) to 3 (fully susceptible) reported by Bai et al. (2008), was used for scoring.Barsandstandard errorsrefer to 11 T2(?)_a plants, 10 T2(?)_b plants, 9 T2(-) plants, 10 Slmlo1 plants and 10 MM plants.