Fungal X-Intrinsic Protein Aquaporin from Trichoderma atroviride: Structural and Functional Considerations

The major intrinsic protein (MIP) superfamily is a key part of the fungal transmembrane transport network. It facilitates the transport of water and low molecular weight solutes across biomembranes. The fungal uncharacterized X-Intrinsic Protein (XIP) subfamily includes the full protein diversity of MIP. Their biological functions still remain fully hypothetical. The aim of this study is still to deepen the diversity and the structure of the XIP subfamily in light of the MIP counterparts-the aquaporins (AQPs) and aquaglyceroporins (AQGPs)-and to describe for the first time their function in the development, biomass accumulation, and mycoparasitic aptitudes of the fungal bioagent Trichoderma atroviride. The fungus-XIP Glade, with one member (TriatXIP), is one of the three clades of MIPs that make up the diversity of T. atroviride MIPs, along with the AQPs (three members) and the AQGPs (three members). TriatXIP resembles those of strict aquaporins, predicting water diffusion and possibly other small polar solutes due to particularly wider ar/R constriction with a Lysine substitution at the LE2 position. The XIP loss of function in Delta TriatXIP mutants slightly delays biomass accumulation but does not impact mycoparasitic activities. Delta TriatMIP forms colonies similar to wild type; however, the hyphae are slightly thinner and colonies produce rare chlamydospores in PDA and specific media, most of which are relatively small and exhibit abnormal morphologies. To better understand the molecular causes of these deviant phenotypes, a wide-metabolic survey of the ATriatXIPs demonstrates that the delayed growth kinetic, correlated to a decrease in respiration rate, is caused by perturbations in the pentose phosphate pathway. Furthermore, the null expression of the XIP gene strongly impacts the expression of four expressed MIP-encoding genes of T. atroviride, a plausible compensating effect which safeguards the physiological integrity and life cycle of the fungus. This paper offers an overview of the fungal XIP family in the biocontrol agent T. atroviride which will be useful for further functional analysis of this particular MIP subfamily in vegetative growth and the environmental stress response in fungi. Ultimately, these findings have implications for the ecophysiology of Trichoderma spp. in natural, agronomic, and industrial systems

Etude de la relation mycoparasitaire Trichoderma harzianum avec Fusarium solani chez l’Olivier ; caractérisations moléculaires et fonctionnelles des aquaporines chez Trichoderma harzianum

Biological disease control through the use of microorganisms has a great potential for future use in integrated pest management. In a multidisciplinary and fundamental context of molecular physio-phytopathology and to provide solutions for the actors in the olive profession and the consumers, we have been studying the activity of a fungal biocontrol agent, Trichoderma harzianum (strain Ths97) against the olive tree pathogen Fusarium solani (strain Fso14), which causes major problems for olive production in Tunisia and elsewhere. The project consists of two parts. In the first part, we have demonstrated that Ths97 is a biocontrol agent effective against the F. solani Fso14 pathogen. Induction of plant defence responses by Ths97 was shown to be partly responsible for the biocontrol effect. In vitro tests further showed that Ths97 develops mycoparasitic activities towards F. solani Fso14, by forming infection structures such as hyphae windings and wedges, appressoria and papillae. In the second part of the study, we investigated the Major Intrinsic Proteins (MIP) superfamily in the Trichoderma genus. This multigenic family has never been investigated in a hyperparasitic fungal species. Seven MIP members are present in T. harzianum, and are classified into 3 subgroups: AQP, AQGP and XIP. Their three-dimensional structures and their putative involvement in transport of water and certain polyols have been examined. Finally, their transcription profiles were monitored in Ths97 in planta in antagonistic situations and in vitro in a parasitic situation with Fso14 and show that 4 MIP are expressed and regulated differentially during the interaction. Our work has shown that Ths97 must be considered as a biological control agent and biostimulator of plant defences, and that MIPs are involved in the trophic relationships between T. harzianum and the environment. These data contributes to the further development of T. harzianum as an efficient biocontrol agent for sustainable protection of olive trees in Tunisia and around the world.La lutte biologique par utilisation de micro-organismes a indéniablement un potentiel de développement considérable. Dans un contexte multidisciplinaire et fondamental de physio-phytopathologie moléculaire et répondant à d’éminents enjeux appliqués et attendus par les acteurs de la profession oléicole et les consommateurs, nous nous sommes projetés dans l’étude des propriétés intrinsèques d’un agent de biocontrôle fongique, Trichoderma harzianum (souche Ths97) contre l’agent de la fusariose Fusarium solani (souche Fso14), qui sévit sévèrement sur une culture pérenne majeure pour la Tunisie, l’oléiculture. Deux axes de recherche ont été menés. Dans le premier axe, nous avons démontré que Ths97 est un agent de biocontrôle efficace contre la virulence de F. solani Fso14. Cette capacité s’accompagne d’une accumulation des défenses chez le partenaire végétal, des accumulations qui sont d’autant plus fortes quand l’agent bénéfique est en présence du pathogène (événements de priming). De même, des tests in vitro montrent que Ths97développe des activités mycoparasites envers F. solani Fso14, en émettant des structures d’infection classiques tels des enroulements et accolements d’hyphes, des appressoria et des papilles. Quant au second axe d’étude, nous avons étudié la superfamille des perméases Major Intrinsic Proteins (MIP) dans le genre Trichoderma. Cette famille multigénique n’a jamais été étudiée chez un agent fongique hyperparasite. Sept membres MIP sont présents chez T. harzianum, et se classent en 3 sous-groupes, les AQP, les AQGP et les XIP. La modélisation des structures tridimensionnelles et les fonctions putatives de transport pour l’eau et quelques polyols ont été étudiées. Enfin, leurs patrons transcriptionnels ont été suivis chez Ths97 in planta en situation d’antagonisme et in vitro en situation de parasitisme vis-à-vis de Fso14, et montrent que 4 MIP sont exprimées et régulées différentiellement selon que Ths97 est au contact de Fso14 ou pas. Nos travaux ont donc mis en lumière que Ths97 doit être considéré comme un agent biofongicide et biostimulateur de défenses végétales, puis que les MIP seraient impliqués dans les relations trophiques que met en place T. harzianum avec son environnement. Ces données devraient intégrer le développement de procédés plus efficaces et/ou plus durables pour la protection des cultures d’oliviers en Tunisie ainsi qu’à travers le monde

MIP diversity from Trichoderma: Structural considerations and transcriptional modulation during mycoparasitic association with Fusarium solani olive trees

Major intrinsic proteins (MIP) are characterized by a transmembrane pore-type architecture that facilitates transport across biomembranes of water and a variety of low molecular weight solutes. They are found in all parts of life, with remarkable protein diversity. Very little is known about MIP from fungi. And yet, it can legitimately be stated that MIP are pivotal molecular components in the privileged relationships fungi enjoy with plants or soil fauna in various environments. To date, MIP have never been studied in a mycoparasitism situation. In this study, the diversity, expression and functional prediction of MIP from the genus Trichoderma were investigated. Trichoderma spp. genomes have at least seven aquaporin genes. Based on a phylogenetic analysis of the translated sequences, members were assigned to the AQP, AQGP and XIP subfamilies. In in vitro and in planta assays with T. harzianum strain Ths97, expression analyses showed that four genes were constitutively expressed. In a mycoparasitic context with Fusarium solani, the causative agent of fusarium dieback on olive tree roots, these genes were up-regulated. This response is of particular interest in analyzing the MIP promoter cis-regulatory motifs, most of which are involved in various carbon and nitrogen metabolisms. Structural analyses provide new insights into the possible role of structural checkpoints by which these members transport water, H2O2, glycerol and, more generally, linear polyols across the membranes. Taken together, these results provide the first evidence that MIP may play a key role in Trichoderma mycoparasitism lifestyle

Proportion of putative transcription factor binding sites (TFBSs) on the 1.5kb promoter region of the expressed MIP genes from <i>Trichoderma harzianum</i>.

<p>MIP promoter sequences from <i>T</i>. <i>harzianum</i> CBS 226.95 v1.0 (JGI) were used as reference. TFBSs were detected with the "Promoter Database of <i>Saccharomyces cerevisiae</i>" (<a href="http://rulai.cshl.edu/SCPD/" target="_blank">http://rulai.cshl.edu/SCPD/</a>), and biological processes (GO) analyzed using «Uniprot» (<a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org/</a>). TFBS nucleotide sites on 1.5kb of each promoter are detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193760#pone.0193760.s006" target="_blank">S3 Table</a>.</p

Structural analysis of the expressed fungal Fsp-like-90014 MIP.

<p><b>(A)</b> Multiple sequence alignments (MSA) were generated from MIP homologs of different groups by group from various fungi computed with Muscle WS in Jalview, and colored by the Taylor color code. Homologous <i>T</i>. <i>harzianum</i> strains CBS 226.95 of the expressed members from <i>Ths97</i> are indicated by a black arrow before their names. Topology of each type is indicated by a ribbon diagram above the sequences on which the different segments are labeled in blue for those in the inner compartment, and red for the outer compartment. The positions of the residues exposed to the light of the channel are designated by a target symbol formed of three black circles under the MSA. The conservation and consensus sequence are given and marked by blue arrows to indicate the positions at the constriction site. (<b>B)</b> Models out of an I-Tasser computation (after different runs to improve the confidence range) are shown in PyMOL scenes. The C-score (estimating the quality of the prediction) is positive for this model used (Cscore = 1.18), suggesting a good level of confidence in all the predictions (the normal range of C-scores being between −5 and 2). The pore established with "MOLE- 2" is materialized by a grid on which the electrostatic potential calculated by APBS with the PARSE forcefield is reported to compare the physicochemical nature of the channels. (<b>C</b>) Focus on the residues of the pore. A blue arrow indicates the ar/R region. (<b>D</b>) Sidechains of the amino acids constricting the channel after both NPA motifs.</p

Biochemical features of <i>Trichoderma</i> MIP.

<p>(<b>A</b>) Relationship between isoelectric point and molecular weight for <i>Trichoderma</i> spp. MIP clusters. Plot showing isoelectric point versus molecular weight for XIP (X), aquaglyceroporins (X) and aquaporins (X). Subgroups are detailed in the phylogeny in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193760#pone.0193760.g001" target="_blank">Fig 1</a> and in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193760#pone.0193760.s004" target="_blank">S1 Table</a>. Means ± SE according to number of MIP members from each subgroup. (<b>B</b>) Amino acid diversity in NPA boxes and Aromatic/arginine selectivity filters in the different MIP subgroups from <i>Trichoderma</i> spp. Exact ar/R locations on MIP proteins are detailed in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193760#pone.0193760.g003" target="_blank">3A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193760#pone.0193760.g004" target="_blank">4A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193760#pone.0193760.g005" target="_blank">5A</a>.</p

Structural analysis of the expressed fungal "other AQGP"-92358 MIP.

<p><b>(A)</b> Multiple sequence alignments (<b>B)</b> Models out of an I-Tasser computation. The C-score (0.51) is positive, suggesting a good level of confidence in all the predictions. The pore is materialized by a grid on which the electrostatic potential is reported to compare the physicochemical nature of the channels. (<b>C</b>) Focus on the residues of the pore. A blue arrow indicates the ar/R region. (<b>D</b>) Sidechains of the amino acids constricting the channel after both NPA motifs. Technical procedures for each item are detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193760#pone.0193760.g003" target="_blank">Fig 3</a> caption.</p

Structural alignments of MIP to highlight noticeable differences in glycerol facilitators <i>versus</i> standard AQP.

<p><b>(A)</b> Structural alignment of different MIP based on the coordinates of resolved structures made with MulPBA on a narrow but still representative sample of MIP of different classes from different kingdoms. The name of the proteins and their relative pdb code is written with distinctive colors on the left of the alignment, itself colored by the Taylor color code in Jalview. A conservation threshold of 50% is applied to highlight the conservation by groups. From this comparison emerges the particular meaning of the conserved GlFp motif NPARD: the conserved negatively-charged residue aspartate makes a salt bridge with an equally conserved residue at exactly one α-turn from it. This bridge quenches both charges by mutual neutralization, allowing their presence in a quite hydrophobic environment for folding purposes (first quarter of α-6). <b>(B)</b> PyMOL scene of the superimposition results from mulPBA displayed as a wireframe diagram of the main chain colored with respect to the sequence name coloring. The channel is shown as a transparent volume to materialize the localization. The sidechain of the conserved arginine from the NPAR motif is shown as sticks, as also are both charged residues occurring only in the GlFp proteins (light and dark red). A red arrow shows the relative displacement (concomitant with this type of electrostatic bridge within the short α-helix of loop E) responsible, at least in part, for a larger pore aperture at its constriction site. Only the NPA α-helices are shown as transparent colored coils (<b>C)</b> Summary of the superimposition score from mulPBA. <b>(D)</b> Structural alignment of MIP from the <i>T</i>. <i>harzianum</i> strain CBS 226.95 homologous to those expressed from <i>Ths97</i> and based on the coordinates of good quality I-Tasser homology models. The MSA is colored by the Taylor color code in Jalview. On the left, the Newick tree established by mulPBA is given showing the relative proximity of both XIP-488926 and AQP-98742 members on one side, and both "other AQGP"-92358 and Fsp-like 90014 members on the other side. Models are consistent with previous data obtained on experimental structures. A conservation threshold of 50% is also applied to highlight the conservation by groups.</p