The Analysis Of Evolution Of The MLO Gene Family In Rosaceae Plants And The Function Of RgMLO6 And RlMLO7 Genes

Powdery mildews caused by podosphera pannosa is the first major disease in the production of cut rose in the world.It is also one of the main diseases of garden rose and potted rose.MLO gene is a new type of disease resistance gene.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.To investigate the evolution and divergence patterns of the MLO(mildew resistance locus o)gene in Rosaceae plants,the sequences and evolutionary characteristics of the MLO gene family were systematically analysed via bioinformatics methods using genome-wide data from six species.Virus induced gene silencing(VIGS)technology was used to preliminarily analyze the function of candidate genes,namely RgMLO6 and RlMLO7,which were screened from the Rosa odorata var.gigantea plants and Rosa longicuspis Bertal plants in the early stage.The results and main conclusions obtained in this study are as follows:1 Sequence Analysis of MLO gene family in Rosaceae plantsIn this study,using three phylogenetic methods(the neighbour-joining,maximum likelihood,and Bayesian methods),we identified 117 MLO genes from 6Rosaceae species.The results of all three phylogenetic analysis methods supported that these genes were divided into six clades.According to the analysis of conserved motifs,A total of 15 conserved motifs with length between 9-45 amino acids were identifieds and only motif 2 was present in all MLO proteins and had 3 nearly invariant amino acid residues.These findings indicated that motif 2 might be a motif shared by the MLO gene family.The structural features of these genes showed that the variations in sequence length among different species were large,although the lengths and the numbers of exons exhibited high degrees of similarity.Selective pressure analysis showed that the MLO gene family was mainly subjected to purifyingselection in Rosaceae plants,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 significant differences in type I functions among different clades,and functionally divergent sites were also detected.Functional distance analysis showed that clade Ⅴ retained more original functions during evolution,whereas clade Ⅰ possibly played a role through specialized functionality.Moreover,we found that 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 and lays the foundation for relevant follow-up studies.2 The functional analysis of RgMLO6 and RlMLO7 genesVIGS technology was used to preliminarily identify the function of RgMLO6 gene.We selected the successfully constructed vector pTRV2-RgMLO6 to transform Agrobacterium.The function of RgMLO6 gene was verified by grafting accelerated VIGS technology.qPCR analysis showed that the relative expression of RgMLO6 gene in the experimental group plants was only 1/8 of that in the control group.The results showed that VIGS technology achieved a significant down-regulation of the target gene expression.The identification results of resistance level to Podosphera pannosa in Rosa odoratavar.gigantea plant showed that the resistance of the plant treated with VIGS technology to Podosphera pannosa has increased from a highly susceptible to moderately resistant leve.Microscopic observation of the growth of Podosphera pannosa on two groups of plants showed that the pathogens grew faster on the epidermal cells of the young leaves of the control group plants,but slower on the epidermal cells of the young leaves of the experimental group plants.In summary,it is initially proved that the RgMLO6 gene participates in the interaction processbetween plants and Podosphera pannosa and plays an important role in the interaction process.VIGS technology was used to preliminarily identify the function of RlMLO7 gene.We selected the successfully constructed vector pTRV2-RlMLO7 to transform Agrobacterium.The function of RlMLO7 gene was verified by grafting accelerated VIGS technology.qPCR analysis showed that the relative expression of RlMLO7 gene in the experimental group plants was only 1/12 of that in the control group,which suggested that the introduction of pTRV2-RlMLO7 effectively inhibit the expression of RlMLO7 gene.The identification results of resistance level to Podosphera pannosa in Rosa longicuspis Bertal plants showed that the resistance of the plant treated with VIGS technology to Podosphera pannosa has increased from a moderately resistant to highly resistant leve.Microscopic observation of the growth of Podosphera pannosa on two groups of plants showed that the growth of the pathogens on the epidermal cells of the young leaves of the control group plants was slow,with a few conidia and mycelia,while the growth of the pathogens on the epidermal cells of the young leaves of the experimental group plants was severely hindered.In summary,it is initially proved that RlMLO7 gene participates in the interaction process between plants and Podosphera pannosa.The VIGS technology was used to preliminarily identify the functions of RgMLO6 and RlMLO7 genes,which laid the foundation for further research on gene functions and provided important information for the genetic improvement of plants.