Pectin as Carbon Source for Monilinia laxa Exoproteome and Expression Profiles of Related Genes

Abstract

Pectin, as part of the fruit cell wall, can be degraded by brown rot fungi by coordinating the production, secretion, and action of extracellular enzymes. In this study, pectin utilization by the necrotroph Monilinia laxa 8L was studied by in vitro and in silico approaches. A total of 403 genes encoding carbohydrate-active enzymes (CAZymes) were identified, including 38 coding a predicted pectin-degrading activity. Analyzing the differences between M. laxa 8L exoproteomes in media containing glucose and pectin as sole carbon sources, we identified 107 pectin-specific proteins, among them, 64.48% harbor a classical secretory activity, including 42 CAZymes and six pectin-degrading proteins. Analyzing the gene-expression patterns of some pectinase families revealed their possible sequential action in pectin disassembly. We found, in vitro, an early pectin-dependent induction of MlRGAE1, MlPG1, and three members of the rhamnosidase family (MlαRHA2, MlαRHA3, and MlαRHA6) and late response of MlPG2 and MlPNL3. M. laxa 8L has the ability to use both pectin and byproducts as carbon sources, based on a functional pectinolytic machinery encoded in its genome, subjected to pectin-dependent regulation and appropriate secretion mechanisms of these pectinolytic enzymes. Differences in the secretion and transcription profile of M. laxa 8L provided insights into the different mechanisms that contribute to brown rot development.

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

“…Cutinase (MfCUT1) of M. fructicola was an infection marker with early expression since 5 h post-inoculation on petals ( Lee et al, 2010 ) and an essential virulence factor in the absence of wounds ( Lee and Bostock, 2006 ; Lee et al, 2010 ). Previous data and this work show that the M. laxa orthologous protein MlCUT1 (Monilinia_042800) was also early expressed during mycelial growth (6 hpi, in vitro cultures ( Rodríguez-Pires et al, 2020 )). Notably, MlCUT1 was detected at 7 dpi on the weak virulent strain 5L, supporting the conclusion that the delay in the development of virulence symptoms by 5L isolate must be caused by an inadequate pattern of exoproteins at 7 dpi.…”

“…Similar strategies have been described studying secretomes in B. cinerea ( González-Fernández et al, 2014 ) and Pyrenophora teres f. teres ( Ismail and Able, 2016 ). An increase of secreted proteins has been reported in B. cinerea when growing in nutritionally rich media compared to more simple media ( Shah et al, 2009a ; Espino et al, 2010 ) or with complex carbon sources like pectin as has been described in M. laxa ( Shah et al, 2009b ; Rodríguez-Pires et al, 2020 ). The higher percentage of secreted proteins, as well as their possible target function in proteins and structural carbohydrates, supports a specialization of M. laxa exoproteome towards peach degradation.…”

“…Interestingly, the virulent factor MlPG1 was detected in the virulent ones but not in the weak virulent 5L, but this isolate produced MlPG6 which has no defined function in pathogenesis. Although in M. fructicola an over-expression of MfPG1 has been associated with smaller lesions ( Chou et al, 2015 ), upregulation of M. laxa MlPG1 and protein identification had been reported with pectin as carbon source ( Rodríguez-Pires et al, 2020 ). Likewise, PG1 was the most widely identified polygalacturonase in B. cinerea ( Shah et al, 2009a ; Shah et al, 2009b ; Espino et al, 2010 ; Shah et al, 2012 ; González-Fernández et al, 2014 ).…”

“…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.…”