Comprehensive Phylogenetic Analysis Sheds Light on the Diversity and Origin of the MLO Family of Integral Membrane Proteins

Abstract

Mildew resistance Locus O (MLO) proteins are polytopic integral membrane proteins that have long been considered as plant-specific and being primarily involved in plant–powdery mildew interactions. However, research in the past decade has revealed that MLO proteins diverged into a family with several clades whose members are associated with different physiological processes. We provide a largely increased dataset of MLO amino acid sequences, comprising nearly all major land plant lineages. Based on this comprehensive dataset, we defined seven phylogenetic clades and reconstructed the likely evolution of the MLO family in embryophytes. We further identified several MLO peptide motifs that are either conserved in all MLO proteins or confined to one or several clades, supporting the notion that clade-specific diversification of MLO functions is associated with particular sequence motifs. In baker’s yeast, some of these motifs are functionally linked to transmembrane (TM) transport of organic molecules and ions. In addition, we attempted to define the evolutionary origin of the MLO family and found that MLO-like proteins with highly diverse membrane topologies are present in green algae, but also in the distinctly related red algae (Rhodophyta), Amoebozoa, and Chromalveolata. Finally, we discovered several instances of putative fusion events between MLO proteins and different kinds of proteins. Such Rosetta stone-type hybrid proteins might be instructive for future analysis of potential MLO functions. Our findings suggest that MLO is an ancient protein that possibly evolved in unicellular photosynthetic eukaryotes, and consolidated in land plants with a conserved topology, comprising seven TM domains and an intrinsically unstructured C-terminus.

Functional characterization of cucumber (Cucumis sativus L.) Clade V MLO genes

“…The data presented there for the Arabidopsis MLO genes are in agreement with our findings (Additional file 2), as they report that in most tissues the major MLO S-gene AtMLO2 is much higher expressed than the other two clade V MLO genes [42]. Rice has two clade IV MLO genes, OsMLO3 and OsMLO6 [25] , it was found that in most rice tissues OsMLO3 transcription was much higher than OsMLO6 transcription [42]. Based on this finding, the authors conclude that OsMLO3 is likely to be the major clade IV MLO in rice, rather than OsMLO6 .…”

Arabidopsis MLO2 is a negative regulator of sensitivity to extracellular reactive oxygen species

“…The biochemical function of MLO proteins remains unknown (Acevedo‐Garcia et al, ; Kusch et al, ). Here, we demonstrate that clade V MLOs act, in unequal genetic redundancy, as negative regulators of ROS signalling.…”

“…Mildew Locus O ( MLO ) genes code for polytopic integral membrane proteins of unknown biological function (Acevedo‐Garcia, Kusch, & Panstruga, ). Mlo was originally identified in barley ( Hordeum vulgare ) but is conserved in plants in small to medium‐sized gene families (Büschges et al, ; Kusch, Pesch, & Panstruga, ). Loss‐of‐function alleles of specific MLOs confer resistance against powdery mildew disease in various plant species; thus, they are considered negative regulators of plant immunity or alternatively as “susceptibility” genes co‐opted by powdery mildew pathogens to promote pathogenesis (Panstruga, ; van Schie & Takken, ).…”

“…The Arabidopsis thaliana (Arabidopsis) genome contains a 15‐member MLO family. MLO2 (At1g11310), MLO6 (At1g61560), and MLO12 (At2g39200) form a phylogenetic group (clade V MLOs) most similar to barley Mlo (Devoto et al, , Kusch et al, ). These genes cofunction with unequal redundancy in the regulation of immunity against Arabidopsis powdery mildew pathogens such as Golovinomyces orontii .…”

Candidate Susceptibility Genes for Powdery and Downy Mildew in Watermelon and Squash

“…MLO genes are characterized by 7 transmembrane helices, similar to metazoan and fungal G-protein coupled receptors [13]. All the MLO susceptibility genes in clade V share highly conserved domains within transmembrane portions of the protein and harbor a four amino acid long motif at the C-terminus that has been associated with PM interaction [14].…”