Mildew Resistance Locus O Proteins Research Articles

Interplay of EXO70 and MLO proteins modulates trichome cell wall composition and susceptibility to powdery mildew.

Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility.

Comprehensive comparative assessment of the Arabidopsis thaliana MLO2-CALMODULIN2 interaction by various in vitro and in vivo protein-protein interaction assays.

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the fungal disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calmodulin-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT-LW/RR mutant variants in comparison with the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.

The Rubber Tree (Heveae brasiliensis) MLO Protein HbMLO12 Promotes Plant Susceptibility to Sustain Infection by a Powdery Mildew Fungus.

Powdery mildew severely affects several important crops and cash plants. Disruption of mildew resistance locus O (MLO) genes elevates resistance against powdery mildew in several plants. However, whether rubber tree (Heveae brasiliensis) MLO proteins are linked to susceptibility remains unknown, owing to technical limitations in the genetic manipulation of this woody plant. A previous study showed that the H. brasiliensis MLO-like protein HbMLO12 demonstrates high amino acid sequence similarity with the known Arabidopsis MLO protein AtMLO12. In this study, we investigated whether HbMLO12 regulates susceptibility to powdery mildew. H. brasiliensis leaves take up exogenously synthesized double-stranded RNAs (dsRNAs), and foliar application of dsRNA homologous to HbMLO12 gene specifically induces HbMLO12 silencing in H. brasiliensis leaf tissues. Notably, HbMLO12 silencing inhibited fungal infection and elevated the immune response during interaction with the rubber tree powdery mildew fungus. Furthermore, the heterologous expression of HbMLO12 suppressed bacterial flg22- and fungal chitin-induced immune responses and enhanced bacterial infection in Arabidopsis. Our study provides evidence that HbMLO12 contributes to susceptibility to powdery mildew. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Unmasking Mildew Resistance Locus O.

Loss of Mildew Resistance Locus O (MLO) in barley confers durable resistance to powdery mildew fungi, which has led to its wide deployment in agriculture. Although MLO is a susceptibility factor, it has become nearly synonymous with powdery mildew resistance. However, MLO has been recently implicated in colonization by arbuscular mycorrhizal fungi and a fungal endophyte, confirming its importance for biotrophic interactions and in promoting symbiosis. Other MLO proteins are involved in essential sensory processes, particularly fertilization and thigmotropism. We propose external stimulus perception as a common theme in these interactions and consider a unified biochemical role, potentially relating to reactive oxygen species (ROS) and calcium regulation, for MLOs across tissues and processes.

Genome-wide identification and evolutionary analysis of MLO gene family in Rosaceae plants

Mutants lacking wild-type MLO (Mildew resistance Locus O) proteins show broad-spectrum resistance to the powdery mildew fungus, and dysregulated cell death control, with spontaneous cell death in response to developmental or abiotic stimuli. In order to understand the evolution and divergence patterns of the MLO gene family in Rosaceae plants, we analysed systematically genome-wide data from Fragaria vesca, Prunus persica, Prunus mume, Malus domestica, Pyrus bretschneideri and Rubus occidentalis based on bioinformatics methods. Using three phylogenetic methods (the neighbour-joining, maximum likelihood, and Bayesian methods), we identified 117 MLO genes from 6 Rosaceae species. The results of all three phylogenetic analysis methods supported that these genes were divided into six clades. Conserved motif analysis found that only motif 2 was present in all MLO proteins and had 3 nearly invariant amino acid residues. The findings indicated that motif 2 might be shared by the MLO gene family. The structural features of these genes showed large variations in sequence length among different species, although the lengths and the numbers of exons exhibited high degrees of similarity. Selective pressure analysis showed extremely significant differences in all 6 clades, with 2, 1, and 1 site(s) under significant positive selection detected in clades III, IV, and VI, respectively. These positive selection sites were important driving forces for the promotion of the functional differentiation of the MLO genes. Functional divergence analysis showed that the significantly divergent sites were located within the domains of the MLO genes. Functional distance analysis showed that the clade V had more conservative functions and might have retained more original functions during the evolutionary process. However, clade I may have undergone extensive altered functional constraints as a specialised functional role. Moreover, the most original function of the MLO genes in Rosaceae could be related to the evolution of their resistance to powdery mildew, which then gradually evolved into functions such as the regulation of flower development, the control of root morphology, and seed evolution due to the different evolutionary rates after gene duplication. These results provide a theoretical basis for further studies of the molecular evolutionary patterns of the plant MLO gene family.

Arabidopsis mlo3 mutant plants exhibit spontaneous callose deposition and signs of early leaf senescence.

Arabidopsis thaliana mlo3 mutant plants are not affected in pathogen infection phenotypes but-reminiscent of mlo2 mutant plants-exhibit spontaneous callose deposition and signs of early leaf senescence. The family of Mildew resistance Locus O (MLO) proteins is best known for its profound effect on the outcome of powdery mildew infections: when the appropriate MLO protein is absent, the plant is fully resistant to otherwise virulent powdery mildew fungi. However, most members of the MLO protein family remain functionally unexplored. Here, we investigate Arabidopsis thaliana MLO3, the closest relative of AtMLO2, AtMLO6 and AtMLO12, which are the Arabidopsis MLO genes implicated in the powdery mildew interaction. The co-expression network of AtMLO3 suggests association of the gene with plant defense-related processes such as salicylic acid homeostasis. Our extensive analysis shows that mlo3 mutants are unaffected regarding their infection phenotype upon challenge with the powdery mildew fungi Golovinomyces orontii and Erysiphe pisi, the oomycete Hyaloperonospora arabidopsidis, and the bacterial pathogen Pseudomonas syringae (the latter both in terms of basal and systemic acquired resistance), indicating that the protein does not play a major role in the response to any of these pathogens. However, mlo3 genotypes display spontaneous callose deposition as well as signs of early senescence in 6- or 7-week-old rosette leaves in the absence of any pathogen challenge, a phenotype that is reminiscent of mlo2 mutant plants. We hypothesize that de-regulated callose deposition in mlo3 genotypes might be the result of a subtle transient aberration of salicylic acid-jasmonic acid homeostasis during development.

Identification and Expression Analysis of Fragaria Vesca MLO Genes Involved in Interaction with Powdery Mildew (Podosphaera Aphanis)

Strawberry powdery mildew, caused by Podosphaeraaphanis is a major fungal disease that affects strawberry yield and quality. In the model plant species Arabidopsis and the crop plants barley, tomato and pea, the Mildew resistance locus O (MLO) proteins have been found to be required for powdery mildew susceptibility. The present study, based on the sequence of a wild plum (Prunus americana) MLO protein, identified 16 MLO genes within the genome of woodland strawberry, Fragaria vesca and examined their expression pattern in response to powdery mildew infection in three diploid strawberry cultivars. Phylogenetic analysis showed that the FvMLO genes can be classified into six clades. Four FvMLO genes were grouped into clade III, which comprises MLO genes from Arabidopsis, tomato and grapevine that mediate powdery mildew susceptibility. A RNA-seq analysis of two diploid strawberry cultivars, F. vescassp. vesca accession Hawaii 4 (HW) and F. vesca f. semperflorens line “Yellow Wonder 5AF7” (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection showed the expression of 12 out of the 16 FvMLO genes. The comparison of Fragments Per Kilobase of transcript per Million mapped reads (FPKM values) detected by RNA-seq and expression values of qRT-PCR for FvMLO genes showed substantial agreement. The FvMLO3 gene, which was grouped in clade III and orthologous to the Arabidopsis,tomato and grapevine genes, was highly expressed in YW compared to other FvMLO genes across varieties. The results showed that FvMLO genes can be used as potential candidates to engineer powdery mildew resistance in strawberry based on MLO suppression or genome editing.

Genome-wide Identification, Classification and Expression Analysis of the Mildew Resistance Locus O ( MLO ) Gene Family in Sweet Orange ( Citrus sinensis )

ABSTRACT Mildew resistance Locus O (MLO), a gene family specific to plants, plays significant roles in the resistance to powdery mildew (PM) and response to a variety of abiotic stresses, plant growth and development. Despite their importance as barley, rice, wheat, few studies are reported in dicots except Arabidopsis; no global analysis has been performed in the burgeoning model fruit plant sweet orange (Citrus sinensis). The recent release of the genome sequences of C. sinensis provides an opportunity to conduct a comprehensive overview the evolution and features of the MLO gene family in sweet orange. In this study, amount to 14 members of the Citrus sinensis MLO gene (CisMLO) family according to their gene structures, conserved motifs, and similitude among their presumptive Arabidopsis and rice orthologs were identified in silico. Based on these analyses, all CisMLOs were grouped into six clades and expanded partly due to one tandem duplication and two segmental duplication events. Survey of their chromosomal distributions uncovered that 14 CisMLOs are localized across 6 chromosomes. Multiple-sequence alignments showed that 11 of them shared seven highly conserved transmembrane domains (TMs), while all of the sweet orange MLO proteins except CisMLO4/14 had a calmodulin-binding domain for MLO function. Expression analysis demonstrated that the MLO gene family has a diverse tissue-specific expression profiles in the sweet orange development and plays potential critical roles in stress responses. These findings will facilitate further studies of evolutionary pattern and biological functions of MLO genes in sweet orange.

MILDEW RESISTANCE LOCUS O Function in Pollen Tube Reception Is Linked to Its Oligomerization and Subcellular Distribution.

Sexual reproduction in flowering plants requires communication between synergid cells and a tip-elongating pollen tube (PT) for the successful delivery of sperm cells to the embryo sac. The reception of the PT relies on signaling within the synergid cell that ultimately leads to the degeneration of the receptive synergid and PT rupture, releasing the sperm cells for double fertilization. In Arabidopsis (Arabidopsis thaliana), NORTIA, a member of the MILDEW RESISTANCE LOCUS O (MLO) family of proteins, plays a critical role in the communication processes regulating PT reception. In this study, we determined that MLO function in PT reception is dependent on MLO protein localization into a Golgi-associated compartment before PT arrival, indicating that PT-triggered regulation of the synergid secretory system is important for synergid function during pollination. Additionally, a structure-function analysis revealed that MLO homooligomerization, mediated by the amino-terminal region of the protein, and carboxyl-terminal tail identity both contribute to MLO activity during PT reception.

A Systematic View of the MLO Family in Rice Suggests Their Novel Roles in Morphological Development, Diurnal Responses, the Light-Signaling Pathway, and Various Stress Responses.

The Mildew resistance Locus O (MLO) family is unique to plants, containing genes that were initially identified as a susceptibility factor to powdery mildew pathogens. However, little is known about the roles and functional diversity of this family in rice, a model crop plant. The rice genome has 12 potential MLO family members. To achieve systematic functional assignments, we performed a phylogenomic analysis by integrating meta-expression data obtained from public sources of microarray data and real-time expression data into a phylogenic tree. Subsequently, we identified 12 MLO genes with various tissue-preferred patterns, including leaf, root, pollen, and ubiquitous expression. This suggested their functional diversity for morphological agronomic traits. We also used these integrated transcriptome data within a phylogenetic context to estimate the functional redundancy or specificity among OsMLO family members. Here, OsMLO12 showed preferential expression in mature pollen; OsMLO4, in the root tips; OsMLO10, throughout the roots except at the tips; and OsMLO8, expression preferential to the leaves and trinucleate pollen. Of particular interest to us was the diurnal expression of OsMLO1, OsMLO3, and OsMLO8, which indicated that they are potentially significant in responses to environmental changes. In osdxr mutants that show defects in the light response, OsMLO1, OsMLO3, OsMLO8, and four calmodulin genes were down-regulated. This finding provides insight into the novel functions of MLO proteins associated with the light-responsive methylerythritol 4-phosphate pathway. In addition, abiotic stress meta-expression data and real-time expression analysis implied that four and five MLO genes in rice are associated with responses to heat and cold stress, respectively. Upregulation of OsMLO3 by Magnaporthe oryzae infection further suggested that this gene participates in the response to pathogens. Our analysis has produced fundamental information that will enhance future studies of the diverse developmental or physiological phenomena mediated by the MLO family in this model plant system.

멜론 흰가루병균 및 식물 호르몬 처리하에서 MLO 유전자군의 발현검정

Powdery mildew disease caused by Podosphaera xanthii is a major concern for Cucumis melo production worldwide. Knowledge on genetic behavior of the related genes and their modulating phytohormones often offer the most efficient approach to develop resistance against different diseases. Mildew Resistance Locus O (MLO) genes encode proteins with seven transmembrane domains that have significant function in plant resistance to powdery mildew fungus. We collected 14 MLO genes from ‘Melonomics’ database. Multiple sequence analysis of MLO proteins revealed the existence of both evolutionary conserved cysteine and proline residues. Moreover, natural genetic variation in conserved amino acids and their replacement by other amino acids are also observed. Real-time quantitative PCR expression analysis was conducted for the leaf samples of P. xanthii infected and phytohormones (methyl jasmonate and salicylic acid) treated plants in melon ‘SCNU1154’ line. Upon P. xanthii infection using 7 different races, the melon line showed variable disease reactions with respect to spread of infection symptoms and disease severity. Three out of 14 CmMLO genes were up-regulated and 7 were down-regulated in leaf samples in response to all races. The up- or down-regulation of the other 4 CmMLO genes was race-specific. The expression of 14 CmMLO genes under methyl jasmonate and salicylic acid application was also variable. Eleven CmMLO genes were up-regulated under salicylic acid treatment, and 7 were up-regulated under methyl jasmonate treatments in C. melo L. Taken together, these stress-responsive CmMLO genes might be useful resources for the development of powdery mildew disease resistant C. melo L.

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

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.

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

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.

Genome-wide identification of the mildew resistance locus O (MLO) gene family in novel cereal model species Brachypodium distachyon

Powdery mildew (PM) is an important plant fungal disease that adversely affects a broad range of angiosperm species, including grass families such as wheat and barley. The MLO (powdery mildew locus O) protein acts as a negative regulator in PM-resistance. Loss-of-function mutation in MLO shows complete resistance to PM disease. In this study, for the first time we reported MLO gene family members in Brachypodium distachyon, model species for grass. 11 well-conserved BdMLO genes were identified on all five chromosomes with a scattered occurrence rather than clustered. The subcellular localization and topology analyses confirmed that all BdMLO proteins anchored to plasma membrane. The seven trans-membrane (TM) and calmodulin-binding domain (CaMBD) sites were well conserved. Amino acid composition showed that BdMLO proteins were leucine-rich (9.9–13.1 %) except BdMLO5 and BdMLO8, which were alanine-rich (10.0 %) and serine-rich (8.7 %), respectively. In silico functional dissection of cis-acting elements revealed that BdMLOs were associated with mainly hormonal, stress, light response and tissue-specific signaling pathways. Phylogenetic relations of BdMLOs within distinct plant species (Arabidopsis, barley, wheat, maize, rice, tomato, pea, pepper and peach) were evaluated. Also, digital expressions of BdMLOs in drought, cold and pathogen infection conditions revealed stress-responsive MLO genes. Phylogenetic and expression analyses provided preliminary evidence that BdMLO2 could be the best susceptibility gene which may play important role in PM resistance. It was concluded that identification and characterization of MLO gene members in Brachypodium will provide essential knowledge for studying full-scale functional analysis of these genes in grass species.