The antagonistic activity of T11 against D V. T11 reduced the colony growth of VI at a rate of Moreover, T11 growth was also temporarily inhibited by VI, although T11 finally jumped the gap in the medium and overgrew the V. Two sets of genome-wide expression analyses were performed using a Trichoderma microarray of , probes to identify genes involved in mycoparasitism of T. The expression of a total number of genes 0. Out of these genes, genes were identified as being differentially expressed in the OV condition Figure 1A , Table 2 and Supplementary Table S2.
Most of the differential expressions Thus, at least these genes can be associated with mycoparasitism potential. Among them, genes were up-regulated By preliminary analysis, GO terms were assigned to out of the genes. Because of overlapping among several GO terms, we further examined whether the differentially expressed genes were associated with similar GO categories.
The genes with catalytic activity that were most differentially expressed corresponded to oxidoreductases, hydrolases, and ligases. In addition, proteins from the 39 genes for which the GO term was not assigned were subjected to a blastp search. Putative function was predicted for 11 of those 39 genes, whereas no function was found for the remaining 28 proteins Table 2. To overcome problems derived from the automatic annotation overlaps and no GO-term assignation , a blastp search was performed with proteins of the differentially expressed genes. As a result, the functional distribution of such genes within physiological events was recorded separated into three groups: metabolism, cellular processes and signaling, and information storage and processing Supplementary Table S2.
Data revealed a marked up-regulation of the transcripts involved in the primary metabolic processes of carbohydrates 21 CAZyme genes , proteins 10 genes , lipids and fatty acids seven genes , and amino acids four genes. The main hydrolytic activities related to mycoparasitism corresponded to glucanases and peptidases.
Up-regulation of transcripts involved in secondary metabolic processes was also observed, with oxidoreductases and monooxygenases being the most abundant, with seven genes each. Within cellular processes and signaling events, transport 14 genes , defense 6 genes , and signaling 4 genes were up-regulated and were the most represented.
Microarrays data analysis and experimental setup of Trichoderma atroviride T11 against Verticillium dahliae VI. A Venn diagram of differentially expressed genes within the set of microarray data. Gene significance was assigned to more than twofold change FDR: 0. Total number of genes and their subgroups of up- and down-regulated genes are showed for each comparative analysis. The intersection between circles displays the total number of genes up- and down-regulated differently expressed under the overgrowing condition OV.
B Scheme of the experimental growth conditions designed for the microarrays. T11 is drawn in dark gray color and VI is delineated in light gray color. Black oval dots represent the sampling areas that were collected for RNA extraction. Summary of the functional distribution of the differentially expressed genes during the overgrowth of Trichoderma atroviride T11 on Verticillium dahliae VI.
To validate the microarray results, RTqPCR assays were performed using 10 arbitrarily chosen genes within the set of the genes differentially expressed in the OV condition Figure 2. RTqPCR profiles confirmed the results obtained in the microarray analysis for all genes evaluated, the highest expression level of the 10 genes analyzed occurring for those detected in the OV condition Figure 2.
Similar results were obtained when including the NT condition in the analysis data not shown. The hit description and distribution in physiological processes of the two gene sets from both comparisons OV vs. CON and OV vs. Up-regulated hydrolase two genes and transporter three genes activities and down-regulated secondary metabolism activity two genes were observed among 15 genes to which function could be assigned in the OV vs.
CON conditions. Likewise, the 18 genes 13 up- and 5 down-regulated showing differential expression between OV and NV conditions formed a separate group see Supplementary Table S4 in Supplementary Material in which transporter activity was the most widely represented four genes , and two out of these four genes were down-regulated. RTqPCR analyses were performed using, respectively, two and four genes chosen arbitrarily from the two sets of 18 genes of each comparison Supplementary Figure S2.
These six genes five up- and one down-regulated showed expression profiles compatible with a mycoparasitic activity since their highest or lowest expression levels corresponded to the OV condition. Identification in the T. Ct values were referred to the CON condition as a basal reference.
Since proteolysis was one of the overrepresented catalytic activities within the set of genes differently expressed when T11 overgrew VI condition OV , we analyzed this enzymatic activity in the OV, NV, and CON conditions. Value obtained in OV The available T. Two oligonucleotides, which contained a sequence target for Pac I and Nt. Bbv CI enzymes, respectively, were designed over cpa1 sequence to amplify a 1,bp fragment from T. The expression analysis of cpa1 gene in T11 wild-type grown under different media conditions showed that the highest expression occurred in MM supplemented with 0.
This expression had a 5. In order to characterize the cpa1 gene functionally and its potential role on biocontrol of V. Transformants were selected through five rounds of hygromycin resistance on PDA plates, with the fourth round being under non-selective media in order to obtain genetic stability of fungal cells.
From a total number of 62 colonies obtained in the first round of selection, only two, namely cpa1 We analyzed the expression of the cpa1 gene and protease activity in three independent biological cultures for transformants and wild-type as a reference condition Figure 3A,B.
The transformant cpa1 The antifungal activity of T. In these tests, unboiled and boiled protein extracts were considered and hyphal growth from conidia of VI was register at 0, 24, 48, and 72 h Figure 3C,D. Absorbance values recorded at 0 h did not show significant differences among the T. Assays using different quantities of boiled protein extracts from the three T. Unboiled protein extracts from strain cpa1 Additionally, the larger volume of unboiled protein extracts from strain cpa1 Trichoderma atroviride T11 and cpa1- overexpressed strains characterization.
A Relative expression of cpa1 gene transcripts compared with the quantity of the reference gene actin. Tests were performed in triplicate and using, at least, three biological replicates, and the data represent mean values with standard deviations. Verticillium wilt caused by the D V. In this present work we found that T. Moreover, results from dual cultures and membrane assays indicate that mycoparasitism and antibiosis seem to be involved in the T. Also, T11 appears to bear higher antagonistic potential against V. Assays involving T11 and VI on a discontinuous medium showed that release of VOCs, which appears to play a role in communication between both fungi, is also involved in the antagonistic potential of T11 against this pathogen Supplementary Figure S1.
Previous studies have demonstrated the usefulness of Trichoderma HDO microarrays in the analysis of transcriptomic changes in T. In the present study, a Trichoderma microarray was used to analyze transcriptomic changes that might occur when T11 overgrows colonies of VI. The use of an unusual biocontrol target such as V. We have used an experimental design similar to those designed to identify mycoparasitism-related genes in T.
In other studies, liquid cultures and fungal host cell walls were used to identify T. A high percentage This seems to effectively counteract a common objection made to the use of microarrays regarding propensity to false-positive detections at low expression levels due to cross-hybridizations Reithner et al. Thus, these genes would be unequivocally associated with an active antagonism of T11 against VI.
Among them, the GO categories that were significantly overrepresented are congruent with a mycoparasitic strategy. When annotation of the T. The up-regulation of 21 genes encoding CAZymes from all classes and of 10 protease serine, aspartic and metallopeptidases genes in OV is compatible with a mycoparasitic process leading to weakening and hydrolysis of fungal host cell walls, which allows for easier access of T11 to nutrients.
A noteworthy increase in expressed CAZyme genes has also been observed in the reported mycoparasitism of T. By contrast, a down-regulation of several glycosyl hydrolases has been observed in mycoparasitism of T. Also, it has been reported that In our study, we have also observed Figure 2 that some T11 genes were up-regulated in the NT condition and higher expression levels were detected in the OV one.
The expression profiles shown in Figure 2 and Supplementary Figure S2 indicate that the level of expression of some genes was affected at a different extent by the proximity of a fungal colony susceptible or not to be parasitized. This agrees with previous reports on the ability of all Trichoderma spp. In this present work, proteolysis was an overrepresented process in condition OV and a peptidases array was consistently up-regulated in this condition, which is in agreement with the significantly higher protease activity detected in OV compared with that in conditions NV and CON.
Those results are in line with previous studies that indicated a fundamental role of peptidases in mycoparasitism by Trichoderma spp. Seidl et al.
The use of Trichoderma species for the biocontrol of plant diseases has been related mainly to their antagonistic abilities against phytopathogenic fungi and oomycetes Howell, You are more than the car, the money, the house, and the stuff Morales, Marcela The Insurance Source source lvcoxmail. Gonzalez, Jose Guadalupe pepe. Giraldo-Tello, Monica corceles21 hotmail.
In addition to overexpressed peptidases and CAZyme genes, a notable transporter activity was observed in the OV condition, which was due to the up-regulation of a diversity of transporter genes, with members of the MFS family being the most abundant Steindorff et al. The up-regulation of small secreted cysteine-rich proteins SCCPs was expected in the OV condition since these proteins are expanded in the genome of T.
Several genes encoding putative proteins related to defense, including two heat-shock proteins and detoxification responses, were up-regulated in OV. Secondary metabolites enhance mycoparasitism by T. Secondary metabolism is enhanced in the mycoparasitism of T11 on VI. Thus, it would be expected that secondary metabolites up-regulated in the OV condition and able to inhibit the target fungus would be different from those secreted by T11 growing alone, as indicated by results from membrane assays Table 1.
The presence of secondary metabolites in boiled-protein extracts from all T11 strains would explain their significant inhibitory effects against VI considering the absence of protein activities in these samples Figure 3D. The effect of secondary metabolites in the antagonism ability of Trichoderma spp. One of the most outstanding properties of T. In this present work, numerous up-regulated genes related to primary and secondary metabolic processes, categorized within oxidoreductase activity, were detected in the OV condition.
An important set of these genes encoding proteins with binding functions was up-regulated in both T. By contrast, other studies have reported repression of this gene category when T. Two prenyltransferases were also up-regulated in the OV condition, this being compatible with an increase in terpene metabolism.
Fungal terpenes are derived from isopentenyl diphosphate building units which cyclization is catalyzed by prenyltransferases to produce mono-, sesqui- and diterpenes that have different antibiotic potential, as well as triterpenes of which ergosterol is the major component of fungal membranes Liang et al. It should be noted that in the OV condition we also observed the up-regulation of three genes encoding isoflavone reductase IFR -like proteins with NmrA domains that are involved in the production of isoflavones. In plants, isoflavonoid phytoalexins are key factors in evolutionary processes shaping the rhizosphere microbiome Philippot et al.
Genes encoding IFRs were up-regulated in T. In these latter studies, IFR genes were functionally related to secondary metabolic processes thought have been also identified as part of a system controlling nitrogen metabolite repression in several fungi Stammers et al. The up-regulation of NmrA-like IFR-negative regulators in mycoparasitic processes might indicate the inactivation of pathways for non-preferred nitrogen sources due to the availability of a simpler nitrogen source.
In the present work, we generated cpa1 -overexpressed transformants as a proof of concept that the set of genes obtained from the microarray analysis are involved in the antagonism of T. This approach proved to be adequate to analyze genes functionally in T. The selection of the cpa1 gene was based on the overrepresentation of the proteolysis activity in the OV condition confirmed by RTqPCR analysis, as well as on the lack of assigned function in the T.
Studies based on transcriptomic analyses have indicated that proteolysis is a major biological process involved in the mycoparasitism by Trichoderma when overgrowing its host Atanasova et al. The increased protease activity of cpa1 -overexpressed mutants of T. The induction of the cpa1 gene was not only triggered when T11 overgrew VI but also under the sole presence of inactivated VI cell walls.
Studies on the characterization of Trichoderma proteases have proved that they are involved in mycoparasitism processes. For instance, the prb1 gene encoding a basic proteinase was induced by presence of phytopathogenic fungi or their cell walls Geremia et al. Similarly, the expression of four transcripts encoding this same aspartic protease, as well as another aspartic protease together with a serine protease and a M22 metalloprotease of T. In other studies, T-DNA insertional mutagenesis and UV-irradiation were used to identify genes involved in the ability of Trichoderma spp.
The nmp1 gene M35 protease identified in Trichoderma guizhouense plays a role in its antifungal activity against the pathogen Fusarium oxysporum f. Some studies of high-throughput analysis have identified the regulation of carboxypeptidases under mycoparasitism events Monteiro et al. However, carboxypeptidases from the M14 family are poorly characterized proteins although a recent report Nauom et al.
Results in this present study indicate that CPA1 contributes somehow to the hydrolysis of VI cells walls and has a key role in the ability of T11 to successfully inhibit the growth of this fungal pathogen.
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Nevertheless, the mechanism underlying the activity of this protease needs to be fully explored. Mycoparasitism is an ancestral feature in Trichoderma but it is not always displayed in a same way against a given host. Our study provides a further insight in the understanding of the relationships between T. Strain T11 has shown outstanding biocontrol abilities against different strains of D V. Microarray data of T11 overgrowing colonies of V. Better understanding of the molecular mechanisms underlying the T. RH and EM contributed conception and design of the study.
MM-D performed the statistical analysis. All authors contributed to manuscript revision, read and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. National Center for Biotechnology Information , U. Journal List Front Microbiol v. Front Microbiol. Published online May Rafael M. Author information Article notes Copyright and License information Disclaimer. This article was submitted to Fungi and Their Interactions, a section of the journal Frontiers in Microbiology.
Received Feb 25; Accepted May 3. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these terms. Abstract Verticillium dahliae , a vascular-colonizing fungus, causes economically important wilt diseases in many crops, including olive trees. Keywords: Verticillium wilt, mycoparasitism, microarray, carboxypeptidase, secondary metabolism, cpa1 -overexpressed mutants. Introduction The use of Trichoderma species for the biocontrol of plant diseases has been related mainly to their antagonistic abilities against phytopathogenic fungi and oomycetes Howell, Statistical Analyses All data were collected from at least three independent replicates and expressed as the mean values.
Open in a separate window. Values are means of three replicates with the corresponding standard deviation. Transcriptomic Changes in Trichoderma atroviride T11 Induced by Verticillium dahliae VI Two sets of genome-wide expression analyses were performed using a Trichoderma microarray of , probes to identify genes involved in mycoparasitism of T. Table 2 Summary of the functional distribution of the differentially expressed genes during the overgrowth of Trichoderma atroviride T11 on Verticillium dahliae VI.
Probe set Up-regulated Down-regulated Metabolism Carbohydrate 21 2 Lipid and fatty acid 7 1 Protein 10 — Amino acid 4 — Nucleic acid 2 — Secondary 20 2 Energy 5 1 Cellular processes and signaling Transport 14 — Defense 6 — Signaling 4 2 Cell—cell contact and communication 1 1 Detoxification 1 — Cell wall and membrane 2 — Regulation 3 — Post-translation events 3 — Information storage and Processing Transcription 1 — Translation 2 — Unknown function 22 6.
Analysis of cpa1 Gene and Characterization of cpa1 -Overexpressed Transformants in vitro One of the genes in the proteolysis group that was identified as being differentially expressed in condition OV compared with conditions NV, NT, and CON, both in microarray and RTqPCR analyses, was selected for further studies in this present work. Discussion Verticillium wilt caused by the D V. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Click here for additional data file. References Ashburner M. The Gene ontology consortium: gene ontology: tool for the unification of biology. Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism. BMC Genomics 14 : A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding.