Show me the way: rust effector targets in heterologous plant systems. C Lorrain, B Petre, S Duplessis. Current opinion in microbiology 46, 19-25

Abstract

For years, the study of rust fungal effectors has been impeded by the lack of molecular genetic tools in rust pathosystems. The recent use of heterologous plants to perform effector screens (effectoromics)-including effector localisation (cellular targets) and protein interactors (molecular targets) in plant cells-has changed the game. These screens revealed that many candidate effectors from various rust fungi target specific plant cell compartments, including chloroplasts, and associate with specific plant protein complexes. Such information represents unparalleled opportunities to understand how effectors sustain extreme parasitic interactions and obligate biotrophy. Despite their limitations, we here portray how the use of heterologous expression systems has been essential for gaining new insight into rust effectors.

Bacterial-Fungal Interactions: ecology, mechanisms and challenges A Deveau, G Bonito, J Uehling, M Paoletti, M Becker, S Bindschedler, … FEMS microbiology reviews

Abstract

Enzyme activities of two recombinant heme-including peroxidases TvDyP1 and TvVP2 identified from the secretome of Trametes versicolor S Amara, T Perrot, D Navarro, A Deroy, A Benkhelfallah, A Chalak, … Applied and Environmental Microbiology, AEM. 02826-17

ABSTRACT

Trametes versicolor is a wood inhabiting Agaricomycete known for its ability to cause strong white rot decay on hardwood and for its high tolerance toward phenolic compounds. The goal of the present work was to give insights on the molecular biology and biochemistry of heme-including class-II and dye-decolorizing peroxidases secreted from this fungus. Proteomic analysis of the secretome of T. versicolor BRFM1218 grown on oak wood revealed a set of 200 secreted proteins among which were a dye-decolorizing peroxidase TvDyP1 and a versatile peroxidase TvVP2. Both peroxidases were heterologously produced in E. coli, and were biochemically characterized and tested for their capacity to oxidize complex substrates. Both peroxidases were found to be active against several substrates in acidic conditions, and TvDyP1 was very stable in a relatively large range of pH (pH 2.0 to 6.0) while TvVP2 was more stable at pH 5.0-6.0 only. Thermostability of both enzymes was also tested and TvDyP1 was globally found to be more stable than TvVP2. After 180 min of incubation at T°C ranging from 30°C to 50°C, activities of TvVP2 drastically decreased retaining 10% to 30% of the its initial activity. In the same conditions, TvDyP1 retained 20% to 80% of enzyme activity. The two proteins were catalytically characterized and TvVP2 was shown to accept a wider range of reducing substrates than TvDyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, TvVP2 displaying a more rapid oxidation of the two compounds. They were tested for their potential interest in dye decolourization of five industrial dyes and TvVP2 presented a wider oxidation and decolourization capacity towards the dye substrates than TvDyP1.

IMPORTANCE Trametes versicolor is a wood inhabiting Agaricomycete known for its ability to cause strong white rot decay on hardwood and for its high tolerance toward phenolic compounds. Among white-rot fungi, the basidiomycete T. versicolor has been extensively studied for its efficiency to degrade wood, and specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of T. versicolor, one dye-decoloririzing peroxidase and one versatile peroxidase were biochemically characterized in depth and compare to other fungal peroxidases.

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors. D Lopez, S Ribeiro, P Label, B Fumanal, JS Venisse, A Kohler, … Frontiers in Microbiology 9, 276

Abstract

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

Function and maturation of the Fe–S center in dihydroxyacid dehydratase from Arabidopsis H Gao, T Azam, S Randeniya, J Couturier, N Rouhier, MK Johnson. Journal of Biological Chemistry, jbc. RA117. 001592

Abstract

Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe–S clusters. The best characterized examples are Escherichia coli DHAD, which contains an oxygen-labile [Fe₄S₄] cluster, and spinach DHAD, which contains an oxygen-resistant [Fe₂S₂] cluster. Although the Fe–S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis. Here, using the combination of UV-visible absorption and circular dichroism, resonance Raman and electron paramagnetic resonance, we spectroscopically characterized the Fe–S center in DHAD from Arabidopsis thaliana (At). Our results indicated that AtDHAD can accommodate [Fe₂S₂] and [Fe₄S₄] clusters. However, only the [Fe₂S₂] cluster–bound form is catalytically active. We found that the [Fe₂S₂] cluster is coordinated by at least one non-cysteinyl ligand, which can be replaced by the thiol group(s) of dithiothreitol.In vitro cluster transfer and reconstitution reactions revealed that [Fe₂S₂] cluster–containing NFU2 protein is likely the physiological cluster donor for in vivo maturation of AtDHAD. In summary, AtDHAD binds either one [Fe₄S4] or one [Fe₂S₂] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [Fe₂S₂] cluster donor for DHAD maturation. This work represents the first in vitro characterization of recombinant AtDHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe–S center in a plant DHAD.