Powdery mildew resistance in the Japanese domestic tobacco cultivar Kokubu is associated with aberrant splicing of MLO orthologues

Abstract

The Japanese domestic tobacco cultivar Kokubu (syn. Kou-fan) is highly resistant to powdery mildew, and this trait is controlled by recessive alleles at two loci. To show the molecular mechanism of powdery mildew resistance of Kokubu, the transcripts and genomic sequences of tobacco Mildew resistance locus O (MLO) orthologues were compared between powdery mildew-resistant and -susceptible cultivars. Two MLO orthologues (NtMLO1 and NtMLO2) corresponding to powdery mildew susceptibility were expressed in tobacco leaves. However, the transcripts of both NtMLO genes in the powdery mildew-resistant cultivars harboured partial deletions or insertions. In a genomic DNA sequence alignment analysis, mutations in intron regions were detected in both NtMLO genes of powdery mildew-resistant cultivars, and these mutations were linked to the powdery mildew-resistant phenotype. Transgenic Kokubu expressing wildtype NtMLO1 or NtMLO2 exhibited severe disease symptoms, the same as those in susceptible cultivars. These results indicate that mutations of intron regions of two NtMLO genes in powdery mildew resistant cultivar Kokubu trigger the aberrant splicing of MLO transcripts and the subsequent inhibition of functional MLO protein synthesis, and that NtMLO1 and NtMLO2 are functionally redundant.