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Transcriptome Dynamics Underlying Chlamydospore Formation in Trichoderma virens GV29-8.

ABSTRACT: Trichoderma spp. are widely used biocontrol agents which are antagonistic to a variety of plant pathogens. Chlamydospores are a type of propagules produced by many fungi that have thick walls and are highly resistant to adverse environmental conditions. Chlamydospore preparations of Trichoderma spp. can withstand various storage conditions, have a longer shelf life than conidial preparations and have better application potential. However, large-scale production of chlamydospores has proven difficult. To understand the molecular mechanisms governing chlamydospore formation (CF) in Trichoderma fungi, we performed a comprehensive analysis of transcriptome dynamics during CF across 8 different developmental time points, which were divided into 4 stages according to PCA analysis: the mycelium growth stage (S1), early and middle stage of CF (S2), flourishing stage of CF (S3), and late stage of CF and mycelia initial autolysis (S4). 2864, 3206, and 3630 DEGs were screened from S2 vs S1, S3 vs S2, and S4 vs S3, respectively. We then identified the pathways and genes that play important roles in each stage of CF by GO, KEGG, STC and WGCNA analysis. The results showed that DEGs in the S2 vs S1 were mainly enriched in organonitrogen compound metabolism, those in S3 vs S2 were mainly involved in secondary metabolite, cell cycle, and N-glycan biosynthesis, and DEGs in S4 vs S3 were mainly involved in lipid, glycogen, and chitin metabolic processes. We speculated that mycelial assimilation and absorption of exogenous nitrogen in the early growth stage (S1), resulted in subsequent nitrogen deficiency (S2). At the same time, secondary metabolites and active oxygen free radicals released during mycelial growth produced an adverse growth environment. The resulting nitrogen-deficient and toxin enriched medium may stimulate cell differentiation by initiating cell cycle regulation to induce morphological transformation of mycelia into chlamydospores. High expression of genes relating to glycogen, lipid, mannan, and chitin synthetic metabolic pathways during the flourishing (S3) and late stages (S4) of CF may be conducive to energy storage and cell wall construction in chlamydospores. For further verifying the functions of the amino sugar and nucleotide sugar metabolism (tre00520) pathway in the CF of T. virens GV29-8 strain, the chitin synthase gene (TRIVIDRAFT_90152), one key gene of the pathway, was deleted and resulted in the dysplasia of mycelia and an incapability to form normal chlamydospores, which illustrated the pathway affecting the CF of T. virens GV29-8 strain. Our results provide a new perspective for understanding the genetics of biochemical pathways involved in CF of Trichoderma spp.

SUBMITTER: Peng X

PROVIDER: S-EPMC8217873 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn fungi, environmental pH is an important signal for development, and successful host colonization depends on homeostasis. Surprisingly, little is known regarding the role of pH in fungal-fungal interactions. Species of Trichoderma grow as soil saprobes but many are primarily mycotrophic, using other fungi as hosts. Therefore, Trichoderma spp. are studied for their potential in biocontrol of plant diseases. Particularly in alkaline soil, pH is a critical limiting factor for these biofungicides, whose optimal growth pH is 4-6. Gaining an understanding of pH adaptability is an important step in broadening the activity spectrum of these economically important fungi.ResultsWe studied the pH-responsive transcription factor PacC by gene knockout and by introduction of a constitutively active allele (pacCc). ΔpacC mutants exhibited reduced growth at alkaline pH, while pacCc strains grew poorly at acidic pH. In plate confrontation assays ΔpacC mutants showed decreased ability to compete with the plant pathogens Rhizoctonia solani and Sclerotium rolfsii. The pacCc strain exhibited an overgrowth of R. solani that was comparable to the wild type, but was unable to overgrow S. rolfsii. To identify genes whose expression is dependent on pH and pacC, we designed oligonucleotide microarrays from the transcript models of the T. virens genome, and compared the transcriptomes of wild type and mutant cultures exposed to high or low pH. Transcript levels from several functional classes were dependent on pacC, on pH, or on both. Furthermore, the expression of a set of pacC-dependent genes was increased in the constitutively-active pacCc strain, and was pH-independent in some, but not all cases.ConclusionsPacC is important for biocontrol-related antagonism of other fungi by T. virens. As much as 5% of the transcriptome is pH-dependent, and of these genes, some 25% depend on pacC. Secondary metabolite biosynthesis and ion transport are among the relevant gene classes. We suggest that ΔpacC mutants may have lost their full biocontrol potential due to their inability to adapt to alkaline pH, to perceive ambient pH, or both. The results raise the novel possibility of genetically manipulating Trichoderma in order to improve adaptability and biocontrol at alkaline pH.

Project description:Cellulose, a linear polymer of ? 1-4, linked glucose, is the most abundant renewable fraction of plant biomass (lignocellulose). It is synergistically converted to glucose by endoglucanase (EG) cellobiohydrolase (CBH) and ?-glucosidase (BGL) of the cellulase complex. BGL plays a major role in the conversion of randomly cleaved cellooligosaccharides into glucose. As it is well known, Saccharomyces cerevisiae can efficiently convert glucose into ethanol under anaerobic conditions. Therefore, S.cerevisiae was genetically modified with the objective of heterologous extracellular expression of the BGLI gene of Trichoderma virens making it capable of utilizing cellobiose to produce ethanol.The cDNA and a genomic sequence of the BGLI gene of Trichoderma virens was cloned in the yeast expression vector pGAPZ? and separately transformed to Saccharomyces cerevisiae. The size of the BGLI cDNA clone was 1363 bp and the genomic DNA clone contained an additional 76 bp single intron following the first exon. The gene was 90% similar to the DNA sequence and 99% similar to the deduced amino acid sequence of 1,4-?-D-glucosidase of T. atroviride (AC237343.1). The BGLI activity expressed by the recombinant genomic clone was 3.4 times greater (1.7 x 10-3 IU ml-1) than that observed for the cDNA clone (5 x 10-4 IU ml-1). Furthermore, the activity was similar to the activity of locally isolated Trichoderma virens (1.5 x 10-3 IU ml-1). The estimated size of the protein was 52 kDA. In fermentation studies, the maximum ethanol production by the genomic and the cDNA clones were 0.36 g and 0.06 g /g of cellobiose respectively. Molecular docking results indicated that the bare protein and cellobiose-protein complex behave in a similar manner with considerable stability in aqueous medium. The deduced binding site and the binding affinity of the constructed homology model appeared to be reasonable. Moreover, it was identified that the five hydrogen bonds formed between the amino acid residues of BGLI and cellobiose are mainly involved in the integrity of enzyme-substrate association.The BGLI activity was remarkably higher in the genomic DNA clone compared to the cDNA clone. Cellobiose was successfully fermented into ethanol by the recombinant S.cerevisiae genomic DNA clone. It has the potential to be used in the industrial production of ethanol as it is capable of simultaneous saccharification and fermentation of cellobiose. Homology modeling, docking studies and molecular dynamics simulation studies will provide a realistic model for further studies in the modification of active site residues which could be followed by mutation studies to improve the catalytic action of BGLI.

Project description:Combating the action of plant pathogenic microorganisms by antagonistic or mycoparasitic fungi has been announced as an attractive biological alternative to the use of chemical fungicides since more than 20 years, and gains additional importance in current trends to environmentally friendly agriculture. Taxa of the fungal genus Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae) contain prominent examples of such biocontrol agents, because they not only antagonize plant-pathogenic fungi, but are also often rhizosphere competent and can enhance plant growth. Identification of the primary factors that regulate the mycoparasitic behaviour and metabolic activities related to it will therefore allow the full ecological significance of this trait to be explored. We performed the analysis of the genome sequence from two mycoparasitic and rhizosphere competent Trichoderma spp. – T. atroviride and T. virens – and compare it to that of the saprophyte T. reesei. The predicted gene inventory of the T. atroviride and T.virens genome, therefore, points to previously unknown mechanisms operating during biocontrol of plant pathogens. The availability of these genomes provides a unique opportunity to develop a deeper understanding of the processes fundamental to mycoparasitism and its application for the breeding of improved biocontrol strains for plant protection. To investigate the potential role in mycoparasitism, microarrays were used to examine T. virens transcript levels when confronted with a potential prey (the plant pathogen Rhizoctonia solani) before contact, during first physical contact and during overgrowth of the host. The study presented here is the result of this analysis. Two biological pools by condition against a common reference control each sample hybridized in dye switch. On the two biological replicates we apply on the pretreated results the linear modeling approach implemented by lmFit and the empirical Bayes statistics implemented by eBayes from the limma R package (Smyth 2004). For mycoparasitism confrontation assays T. virens was grown on potato dextrose agar plates (BD Dicfo, Franklin Lakes, NJ, USA), covered with cellophane, in constant light at 25°C and harvested when the mycelia were ca. 5 mm apart (before contact), at contact of the mycelia and after T. virens had overgrown the host fungus Rhizoctonia solani by ca. 5 mm (after contact). As control T. virens was confronted with itself and harvested at contact. Peripheral hyphal zones from each confrontation stage were harvested and shock frozen in liquid nitrogen. Mycelia were ground to a fine powder under liquid nitrogen and total RNA was isolated using the guanidinium thiocyanate method (Sambrook, 2001).

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Transcriptome Dynamics Underlying Chlamydospore Formation in Trichoderma virens GV29-8.

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