Pectin methyl esterases and rhamnogalacturonan hydrolases: weapons for successful Monilinia laxa infection in stone fruit?

Abstract

The secretion of cell wall‐degrading enzymes is one of the mechanisms used by necrotrophic fungi to colonize host tissues. However, information about virulence factors of Monilinia spp., the causal agents of brown rot in stone fruit, is scarce. Plant cell walls have three main components that are broken down by fungal enzymes: cellulose, hemicellulose and pectin. In order to identify Monilinia laxa candidate proteins involved in pectin hydrolysis, two in vitro approaches were conducted: (i) phenotypic and ecophysiological characterization of growth of the pathogen at different pHs, in glucose‐ and pectin‐containing solid media for 7 days' incubation; and (ii) expression analysis of genes encoding M. laxa pectin methyl esterases (MlPMEs) and rhamnogalacturonan hydrolases (MlRG‐HYDs) after incubation for 0.5, 2, 6, 24 and 48 h in glucose‐ and pectin‐containing liquid media. Phenotypic tests showed the role of carbon source on M. laxa growth rate and aggressiveness, and indicated that pectinases were greatly affected by pH. Gene expression analyses uncovered differences among members of each family of pectinases and between the two families, defining sets of genes expressed at earlier (0.5–6 h) and later (48 h) phases. Notably, the up‐ or down‐regulation of these target genes was carbon source‐dependent. Finally, an in vivo study confirmed the synergistic and complementary role that these genes play in the M. laxa–stone fruit pathosystem. Based on these results, it is hypothesized that MlPME2, MlRG‐HYD1 and MlRG‐HYD2 may be potential virulence factors of M. laxa in the process from infection to colonization.

Depicting the battle between nectarine and Monilinia laxa: the fruit developmental stage dictates the effectiveness of the host defenses and the pathogen’s infection strategies

“…on culture media has been previously reported 11 . A rhamnogalacturonan hydrolase (glycoside hydrolase family 28, Monilinia_041700 ), which was highly expressed at both 24 and 48 hpi, was already described as a putative virulence factor in M. laxa infecting peaches 43 .…”

“…The M. laxa single-spore strain 8L (ML8L, Spanish Culture Type Collection number CECT 21100) was used for all experiments. Fungal conidial suspensions were maintained and prepared, as described by Baró-Montel et al 43 .…”

“…Frozen samples were ground using a mortar and pestle. The total RNA was extracted following the protocol described previously 43 . Contaminant DNA was removed by treating RNA extracts with Turbo DNA-free DNase (Ambion, TX, USA).…”

Proteomic Studies to Understand the Mechanisms of Peach Tissue Degradation by Monilinia laxa

“…Among pectin lyases (PNLs), MlPNL1 was not detected, MlPNL3 was only identified in 5L, while the occurrence of MlPNL2 was in all of them. Pectin methyl esterases played a role in de-esterification in pectin breakdown, and particular MlPME2 had been described as a possible virulence factor in fruit ( Baró-Montel et al, 2019 ), and highly expressed concerning other Monilinia spp ( De Miccolis Angelini et al, 2018 ). The presence of some PME seems to be constitutive when M. laxa grew in pectin or glucose culture ( Rodríguez-Pires et al, 2020 ), but here MlPME3 was absent in the weak virulent 5L.…”

“…Accordingly, the polygalacturonase family ( Chou et al, 2015 ) and MfCUT1 cutinase ( Lee et al, 2010 ) have also captured significant attention due to their essential roles in the pathogenesis of M. fructicola . Polygalacturonase, pectin and pectate lyase, rhamnogalacturonan acetyl esterase, rhamnogalacturonan hydrolase, and α- l -rhamnosidase gene families related to pectin degradation have been identified from M. laxa ( Baró-Montel et al, 2019 ; Rodríguez-Pires et al, 2020 ). The application of transcriptomics to Monilinia spp.…”