Expression of Five Endopolygalacturonase Genes and Demonstration that MfPG1 Overexpression Diminishes Virulence in the Brown Rot Pathogen Monilinia fructicola

Abstract

Monilinia fructicola is a devastating pathogen on stone fruits, causing blossom blight and fruit rot. Little is known about pathogenic mechanisms in M. fructicola and related Monilinia species. In this study, five endopolygalacturonase (endo-PG) genes were cloned and functionally characterized in M. fructicola. Quantitative reverse-transcriptase PCR (qRT-PCR) revealed that the five MfPG genes are differentially expressed during pathogenesis and in culture under various pH regimes and carbon and nitrogen sources. MfPG1 encodes the major endo-PG and is expressed to significantly higher levels compared to the other four MfPGs in culture and in planta. MfPG1 function during pathogenesis was evaluated by examining the disease phenotypes and gene expression patterns in M. fructicola MfPG1-overexpressing strains and in strains carrying the β-glucuronidase (GUS) reporter gene fused with MfPG1 (MfPG1-GUS). The MFPG1-GUS reporter was expressed in situ in conidia and hyphae following inoculation of flower petals, and qRT-PCR analysis confirmed MfPG1 expression during pathogenesis. MfPG1-overexpressing strains produced smaller lesions and higher levels of reactive oxygen species (ROS) on the petals of peach and rose flowers than the wild-type strain, suggesting that MfPG1 affecting fungal virulence might be in part resulted from the increase of ROS in the Prunus–M. fructicola interactions.

Epidemiología y control de la podredumbre parda del melocotón en postcosecha

“…Pectin-lyases and polygalacturonases are two of the extracellular enzymes required to degrade this complex set of polysaccharides, of these two types of enzymes polygalactoronases had been studied in depth due to their relation with virulence. M. fructicola has 5 genes coding for endopolygalacturonase enzymes, and a strain overexpressing MfPG1, the most highly expresed of them, produced smaller lesions and an increase in reactive oxygen species on the petals of peach and rose flowers, indicating a virulence function (Chou et al, 2015). The relative expression of MfPG2 and MfPG3 increased 12-fold and 6-fold, respectively, when the ambient pH was lowered from 4.5 to 3.6 suggesting the importance of ambient pH for the secretion of pathogenicity factors by M. fructicola (De Cal et al, 2013).…”

“…But recently thanks to new technologies and molecular tools available: DNA sequencing, fungal transformation and proteomics techniques, a more in depth study of the extracellular degrading enzymes secreted by fungal pathogens have been done, opening a new avenue of enzymatic studies (Chou et al, 2015;González-Fernández et al, 2014;Lee et al, 2010;Li et al, 2004). …”

“…More recent studies, revealed that MFCUT1 the main cutinase of M. fructicola is a virulence factor, required to penetrate plant tissues (Lee and Bostock, 2006a;Lee et al, 2010). Additionally 5 different endopolygalacturonases, have been detected and the most abundantly expresed of them, MfPG1 has an impact in M. fructicola virulence (Chou et al, 2015). These last developments have been possible due to genomic advances (M. fructicola genome sequencing and Agrobacterium tumefaciens-and protoplast CaCl2/poly ethylene glycol-mediated transformation of M. fructicola) (Chou et al, 2015;Lee and Bostock, 2006a).…”

“…Additionally 5 different endopolygalacturonases, have been detected and the most abundantly expresed of them, MfPG1 has an impact in M. fructicola virulence (Chou et al, 2015). These last developments have been possible due to genomic advances (M. fructicola genome sequencing and Agrobacterium tumefaciens-and protoplast CaCl2/poly ethylene glycol-mediated transformation of M. fructicola) (Chou et al, 2015;Lee and Bostock, 2006a). However, M. fructigena and M. laxa genomes are still unsequenced, and the one from M. fructicola is not readily available to the scientific community, difficulting further advancements through this avenue.…”

“…Agrobacterium tumefaciens-mediated transformation (Chen et al, 2017;Dai et al, 2003;Lee and Bostock, 2006a;Lee et al, 2010) but also by CaCl2/poly ethylene glycol-mediated transformation (Chou et al, 2015). However, the yield of transformants in these studies was low and there are no studies of M. laxa transformation.…”