Hello everyone, the article we are tweeting today is from Biomolecules
“Endo-β-1,3-glucanase (GH16 Family) from
Trichoder
ma harzianumParticipates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens" by Marcela Suriani Ribeiro et al. of the Institute of Biological Sciences of the Federal University of Brazil.
Trichoderma is known for its ability to produce lysozing enzymes such as exonuclease, endonuclease, chitinases, and proteases, which play an important role in cell wall degradation of plant pathogens. β-dextranase plays a vital role in the morphogenesis-morphology of Trichoderma species development and differentiation, in which β-dextran is the main component of its cell wall. Despite the importance of dextranase in the parasitization process of Trichoderma fungi, there are few studies on functional analysis of dextranase. In the study, the authors studied the functional role of the gluc31 gene, which encodes the endo-β-1,3-glucanase of the All16 family of Trichoderma hastellois. The authors found that the deletion of the gluc31 gene did not affect the in vivo fungal parasitic capacity of the mutant ALL42; however, gluc31 significantly affected cell wall tissue. Polymer measurements and fluorescence microscopy analysis showed that the deletion of the gluc31 gene induced a compensatory response by increasing the production of chitin and dextran polymers on the wall of mutant hyphae cells. Mutant strains are more resistant to the fungicide benzyl chloride compared to parental strains. In addition, qRT-PCR analysis showed that the loss of gluc31 in rice led to differences in other glycosyl hydrolase expressions of the GH16 family due to functional redundancy between dextranases.
Gluc31 is involved in cell wall remodeling of Hastenella
The effect of Gluc31 on the cell wall of Hazelella was evaluated by measuring dextran and chitin content. A comparison of dextran and chitin content in mutants (∆glu31) and wild-type (ALL42) strains showed that the mutant strains (∆glu31) had certain high levels of N-acetyl-glucosamine and β-1, 3-dextran content (Figures 2F, H). Fluorescence microscopy analysis showed that Gluc31 was involved in cell wall dynamics. Calcium-fluoride-impregnated hyphae have a large accumulation of chitin in the apical hyphae (Figure 2D). In addition, the deletion of Gluc31 reduces hyphae width (Figures 3A, C) and increases cell wall thickness (Figures 3B, D). The findings suggest that Gluc31 is involved in cell wall remodeling as a compensatory effect, promoting the accumulation of chitin and dextran in response to the deletion of the Gluc31 gene.
Cell viability assay
Since chitin and dextran levels are affected by the deletion of the gluc31 gene, the authors tested the antifungal resistance of the parentAL ALL42 and ∆gluc31 strains. PDA plate experiments have shown that the ∆gluc31 mutant strain has better intrinsic growth capacity in the presence of 0.5 μg/mL of benzyl methyl compared to ALL42 parental strains (Figure 4A). This effect was assessed over a 6-day growth period and the results showed that from day 4 of growth, the mutant strain had significant resistance to benzylmetal compared to the ALL42 parental strain (Figure 4B). In addition, experiments have investigated the effect of benzylamine on the viability of parental ALL42 and ∆gluc31 strains in benzyl liquid medium, ∆ gluc31 strains are more resistant than parental strain ALL42 (Figure 4C), similar to those seen in macroscopic experiments (Figures 4A, B). At 0.5 μg/mL of benzyl, the growth rate of the ∆gluc31 mutant strain began to decrease, but remained higher than that of the parental strain ALL42. In addition, the parental strain also grew at a low rate in 1.5 μg/mL of benzyl compared to the ∆gluc31 strain (Figure 4C). Taken together, the results showed that the ∆gluc31 mutant strain was more resistant to the fungicide benzyl treatment, possibly due to the high accumulation of chitin and dextran in the cell wall of the mutant strain (Figure 2).
β-glucanase encoded by the gluc31 gene does not require its potential antagonism
By gene expression analysis, the activity of the gluc31 gene in the antagonistic process was verified (Figures 5A, B). There is differential expression of the gluc31 gene in the confrontation of Hashiella with plant pathogens. Gluc31 gene expression is upregulated during the antagonism of Hassyella with plant pathogens and sclerotia under contact conditions (Figure 5C). On the other hand, under the same conditions, Gluc31 was not regulated during contact between Hassporium and Tribulus. In addition, under post-exposure conditions, the expression of gluc31 remained unchanged when hazillain and solan antagonized, while under the same conditions, the expression of gluc31 was down-regulated when sclerotium orchids were exposed. In addition, gluc31 levels were down-regulated during the confrontation with Trisporum compared to contact conditions (Figure 5D). Using the scale described by Bell et al., there was no significant difference in antagonistic efficiency between wild-type (ALL42) and mutant (∆glu31) strains and plant pathogens. Combined with statistical analysis, the above results show that Gluc31 does not need to fight plant pathogens.
Verifying that Gluc31 is involved in compensatory reactions during fungal parasitism, the authors evaluated gene expression cultures of glucose enzymes, β-N-acetyl glucosamine and lichenase, as well as chitin synthase and glucan synthase cultures ALL42 and ∆ gluc31 after exposure to the cell wall of Solan (Figure 6). After incubation for 48 h, no difference in chitinase activity was observed, but a significant increase in NAGase activity was observed ∆ Gluc31 strain, as well as the upregulation of the expression of chitin synthase (~40-fold) relative to wild-type ALL42, thus indicating that the deletion of Gluc31 did not affect fungal parasitism, but affected cell wall remodeling (Figure 2A-C) as expected, ∆ gluc31 strains had lower total β-1,3-dextran activity than the ALL42 strain (Figure 6D). In addition, the activity of lichenase (an enzyme-catalyzed hydrolysis of (1->4)-β-d glucoside bonds β-d-glucan containing (1->3) bonds and (1->4) bonds) decreased and dextran synthase levels were upregulated (~40-fold) ∆ gluuc31 strains compared to the parent ALL42 strains (Figure 6f). The findings confirm that even during fungal parasitism, the absence of Gluc31 compensates for cell wall remodeling.
Effects on gene expression of the GH16 family in ∆ Gluc31 strains
To analyze the effect of gluc31 gene deletion in the fungal parasitic process, the authors conducted a direct confrontation test on plant pathogens in a Petri dish. The results showed that 7 of these 14 genes were upregulated when expressed when exposed to contact. In contrast, the GH16 gene of the ∆gluc31 strain was downregulated during confrontation, but the 84167 gene was upregulated relative to the WT strain, and the genes 15000, 503986, and 513340 were expressed the most under contact conditions (14-fold, 6-fold, and 15-fold, respectively) in WT strains. However, the expression levels of these genes in the ∆gluc31 mutant strains did not change, suggesting that they play an important role in the antagonism of Nightshade plants. Interestingly, most of the induced GH16 genes are grouped by homology in WT (Figure 8), suggesting that this subpopulation may play a role in the interaction of fungal parasites. The authors' findings suggest that the absence of gluc31 leads to inhibition of the glucose breakdown system.
Based on the discovery of the gluc31 gene, as well as previous studies of fungi β-1,3-glucanase, the authors propose a functional gene study based on homologous recombinant knockout techniques. Although gluc31 exists in the process of antagonism against various plant pathogens, it has not been found to play a key role in this process. In addition, the observed characteristics of the mutant strain are thought to be common to the deletion of fungal cell wall-related genes, thus suggesting their hypothetical function in cell wall dynamics, as confirmed by the results of gluc31 functional analysis in vitro. This study provides a different perspective on the activity of β-1, 3-glucanase, and the role of these enzymes in the parasitism of Trichoderma fungi has been widely recognized.