Functional analyses of four rose MLO genes (RhMLO1-4) in homologous and heterologous expression systems

Powdery mildew, which is caused by fungi belonging to the order Erysiphales, is one of the most common plant diseases worldwide. Every year, people spend a lot of money on fungicides and disease-resistant varieties to control powdery mildew. However, to move towards green, clean, sustainable agriculture, scientist must create plant cultivars with nature disease resistance to ensure user safety by avoiding fungicides as far as possible. Scientists have successfully identified the Mildew resistance Locus O (MLO) genes, which confer susceptibility to powdery mildew a fungal disease caused by various species of the genus Erysiphe. In this study, we focused on four previously identified rose MLO genes (RhMLO1 to RhMLO4) as putative candidates for powdery mildew susceptibility, in order to determine the function of each of these paralogues. By studying the transient Agrobacterium-mediated transformation of the petals of 96 different cut rose cultivars, we partly determined the genetic complexity of transient gene expression in rose petal cells, which is prerequisite to successfully transfer the disease-resistant MLO genes into rose plants. Inoculation experiments involving different powdery mildews (Podosphaera pannosa and Oidium neolycopersici), conducted after the infiltration of transiently expressed RhMLO genes into the petals of the ‘Sebastian Kneipp’ rose cultivar and the ‘Kokubu’ tobacco mutant via Agrobacterium, concluded that the transient inoculation of rose petals and tobacco mutant leaves had not increased the effect of the infiltrated areas. Therefore, a more in-depth experiment was needed to demonstrate the susceptibility of rose MLO genes on powdery mildew susceptibility of plant tissues. Therefore, we overexpressed rose MLO genes in a MLO triple mutant of Arabidopsis cultivar Col-0 (Atmlo2/6/12), which lacked any of the three functional Arabidopsis MLO genes and therefore was completely resistant to powdery mildew, using the flower dip method. The results showed that two of the four rose MLO genes (RhMLO1 and RhMLO2) increased susceptibility to powdery mildew when transformed into Atmlo2/6/12, partially restoring the ability to be infected by the pathogenic fungus Podosphaera xanthii. However, the other two genes, RhMLO3 and RhMLO4, showed no change in (recovery of) powdery mildew susceptibility. These are genes could be candidates for other functions in roses or they might have a role in combination with the two active RhMLO genes which might be analysed in future studies.