Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Research community interested in the role of Trichodema in the rhizosphere of host plants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student (PhD) and postdoctoral researcher were involved in aspects of this research. The graduate student completed his degree during the time covered by this report, and is employed in the biotechnology industry developing a vaccine for COVID-19. Both were active members of our weekly laboratory meetings (my laboratory and a colleague's group) where they presented their current research findings or a recent published article relevant to our research. Both have collaborated with other laboratories on campus as well as the ARS group in College Station. The collaborations involved identification of secondary metabolites or proteins through HPLC, MS/MS, and NMR. Both have received training through Texas A&M System for working in a BSL-2 laboratory and required training in discrimination and research and institutional situations. How have the results been disseminated to communities of interest?We have published two papers. Both the graduate student and postdoctoral researcher presented departmental seminars on their research efforts. In addition, we have held zoom meetings with our colleague in Israel and his research group. What do you plan to do during the next reporting period to accomplish the goals?We anticipate submitting two additional manuscripts this next reporting period. One will address a unique hydrophobin from T. virens that we have characterized for its role in root colonization and induced systemic resistance. The second manuscript will report our findings on identifying HUB genes by computational approaches.
Impacts
What was accomplished under these goals?
A previous transcriptomic analysis of the interaction of maize roots and T. virens identified several differentially-expressed genes (DEGs) of T. virens that were significantly upregulated in the presence of the roots. One of the genes is Tv_51662, which codes for a hypothetical protein predicted to be a salicylic acid monooxygenase (SAM), which degrades salicylic acid (SA) to catechol. As expression of Tv_51662 was only detectable in T. virens when grown with maize, we hypothesized that this specific SAM gene may play a major role in regulating interactions between T. virens and plant host roots. More specifically, we predicted that this SAM may play a major role in degrading SA to dampen host defenses to facilitate root colonization. Compared to several other SAM genes (Tv_223757, Tv_29989, Tv_147231, Tv_228034, and Tv_76629), Tv_51662 was the only SAM gene that was significantly overexpressed in the presence of maize. We subsequently generated several Δ51662 knockout mutants of T. virens. Compared to WT, these mutants displayed no changes in radial growth on minimal media (VMS), but demonstrated reduced growth when grown on VMS supplemented with SA (1mM). We also grew WT and Δ51662 mutants under hydroponic conditions with maize seedlings and extracted root hormones and metabolites for LC-MS/MS analysis. The preliminary results of this experiment showed that SA levels were highest in untreated maize roots and lowest with roots treated with WT T. virens. Roots treated with Δ51662 had SA levels that were between untreated and WT suggesting that Tv_51662 does play a significantly important role in controlling host SA levels. We subsequently examined root colonization by Δ51662. Compared to WT, the Δ51662 mutants demonstrated significantly reduced levels of root colonization. Most surprisingly, the Δ51662 mutants were no longer able to trigger ISR in treated maize, as foliar lesions caused by Colletotrichum graminicola were much larger in size compared to those treated with WT T. virens. Currently, there is work to clone Tv_51662 to express in E. coli in order to confirm its function in degrading SA in vitro. Using the same transcriptomic data, we employed a different streamlined computational network analysis pipeline (Kim, et al. 2015, Kim, et al. 2018) to investigate the systemic changes across DEGs in the T. virens-maize root interaction. Rather than focusing on read counts from a list of DEGs, this computational approach provides a mechanism to make meaningful predictions from the generated datasets. Based on this analysis of the transcriptomic data (WT with and without roots), several candidate hub genes were identified that may play significant roles in regulating interactions between T. virens and maize. Of the eight candidate genes, we arbitrarily chose three genes for further characterization to confirm the validity of our approach. The first gene, TRIVIDRAFT_18067, encodes for a hypothetical protein that may be an extracellular matrix protein. The knockout mutants exhibited no significant morphological changes, with radial growth, germination, conidiation, direct antagonism of the fungal pathogens Rhizoctonia solani or Pythium ultimum, and maize root colonization being indistinguishable from that of WT. Surprisingly, the mutants were no longer able to trigger ISR in associated maize plants. The second gene, TRIVIDRAFT_72259, encodes for a hypothetical protein and predicted to be a gluconolactonase. Knockout mutants of TRIVIDRAFT_72259 displayed no different phenotypic changes, antagonism towards R. solani and P. ultimum, or ISR compared to WT. However, they were significantly reduced in their ability to colonize maize roots compared to WT. The third gene, TRIVIDRAFT_75844, encodes for a small GTPase Rho3. Unlike the other two mutants, knockout mutants of TRIVIDRAFT_75844 displayed drastically reduced radial growth, germination, conidiation, direct antagonism of fungal pathogens R. solani and P. ultimum, and root colonization compared to WT. The only characteristic assayed that was no altered was the mutants ability to trigger ISR in host maize plants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Wang, K.D., Gorman, Z., Huang, P.C., Kenerley, C.M., and Kolomiets, M.V. Trichoderma virens colonization of maize roots triggers rapid accumulation of 12-oxophytodienoate and two alpha-ketols in leaves as priming agents of induced systemic resistance. Plant Signaling and Behavior. 15. DOI: 10.1080/15592324.2020.1792187
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Taylor, J.T., Mukherjee, P.K., Puckhaber, L.S., Dixit, K., Igumenova, T.I., Suh, C., Horwitz, B.A., and Kenerley, C.M. Deletion of the Trichoderma virens NRPS, Tex7, induces accumulation of the anti-cancer compound heptelidic acid. Biochemical and Biophysical Research Communications. 529: 672-677.
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Progress 05/29/19 to 09/30/19
Outputs
Target Audience:Research community interested in the role of Trichoderma in the rhizosphere of host plants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student has been participated in this project. The student has been an active member of our weekly laboratory meetings where he has the opportunity to present research updates and contribute to paper discussions. As some of his research has involved NMR and MS/MS, he has had the opportunity to interact and collaborate with these advanced facilities on the Texas A&M campus. An undergraduate has also been involved in aspects of the project by assisting in the experiments and presenting her findings at laboratory meetings. How have the results been disseminated to communities of interest?We have published two papers and anticipate the completion of the graduate student's dissertation this year. He will present a Departmental seminar this year, and has monthly interactions with our collaborator in Israel via Zoom. What do you plan to do during the next reporting period to accomplish the goals?We have several pieces of research to conclude, which are directed towards the analysis of selected HUB genes that were identified by the analysis of transcriptomic data of T. virens colonizing maize roots. The research will involve the creation of several mutants that will be assessed for their colonization potential, ability to induce host resistance, and developmental aspects of fungal mycelia. A second set of experiments will test the role of over-expression a novel hydrophobin in mutants for effects on host growth and resistance induction.
Impacts
What was accomplished under these goals?
As most species of Trichoderma are capable of colonizing roots of numerous plants resulting in the activation of host defenses against pathogens, we have sought to define genes involved in the colonization process. One aspect of our focus has been on genes and gene clusters encoding for peptide synthetases in T. virens. Screening a series of core genes in putative peptide synthetases for their expression levels during root colonization of maize resulted in identifying several gene candidates for construction of mutants to further assess their role in root colonization. The knockout mutant in a gene named Tex7 was still capable of root colonization, but inhibited the growth of maize young plants (4 week-old). Testing for general production of secondary metabolites by TLC revealed the appearance of an anomalous compound in large quantities that was not in the wild-type strain. Strains of T. virens has been separated into two different groups based primarily on the production of secondary metabolites. The mutant of Tex7 was constructed in a strain that would have been expected to produce gliotoxin, viridin, viridiol but not heptelidic acid or gliovirin. Following a combination of HPLC, LCMS-MS, and NMR, this compound was surprisingly, identified as heptelidic acid. This discovery alters the strain classification structure and selection process of putative biocontrol strains of T. virens. As hydrophobins produced by fungi have been shown to affect root colonization, we undertook an examination of two class I hydrophobins from T. virens. Deletion mutants of the first hydrophobin (HFB9a) were generated and displayed no differences in growth, mycoparasitism, nor biocontrol capability as compared to wild type. However, the surface hydrophobicity of the mycelium was significantly disrupted in the deletion mutants, and the mutants showed significantly less cellulase and chitinase activity. Plants treated with strains lacking the HFB9a gene were significantly less colonized compared to the WT treated plants. Thus, one hypothesis is that the coordination between the hydrophobin and plant cell wall-degrading enzymes has been altered in the mutant. Additionally, plants treated with the mutant strains were unable to induce systemic resistance (ISR) against the foliar pathogen Colletotrichum graminicola. Recombinant protein was used to complement the enzyme activity in the deletion strains and was found to enhance the activity of commercially sourced chitinase and cellulase. The production of the elicitor SM1 secreted by T. virens in the mutants was compared to the WT by western blot and found to be similar, suggesting that there is another mechanism behind the ISR phenotype. The second class I hydrophobin (HFB3a) produced by T. virens shares high homology with the T. asperellum hydrophobin TasHyd1, which is responsible for attachment of the fungus to plant roots. We generated deletion mutants in this locus and found no changes in growth, mycoparasitism, or biocontrol ability compared to wild type. However, the mutants significantly over-colonized treated plants and demonstrated a lack of ISR against Cochliobolus heterostrophus. We are in the process of developing overexpression strains to determine if colonization and ISR will be enhanced by producing more transcript of this gene. Another study was initiated to access gene expression by wild type T. virens introduced into a hydroponic system containing roots of maize seedlings over a time course of five different time points. RNA was extracted from collected fungal tissues and sequenced by RNA-seq. The reads were analyzed using HISAT2, Stringtie, and EdgeR for read alignment, abundance determination, and differential expression analysis, respectively. This analysis returned a large data set of differentially expressed genes. A computational approach was adopted to enrich the likelihood that a selected target gene would have a high impact on the root colonization process. A gene co-expression network that contains all the genes identified in the RNA-seq analysis and connects the genes together based on the likelihood that the expression between the two would be similar at a given point of time was produced. Then two methods of cluster analysis were used to generate groups of genes based on time and level of expression. The first group was derived from scatter plots with the fold change of two different time points on the x and y axes (eg. 6 hrs vs 12 hrs, 12 hrs vs 15 hrs). These scatter plots were then subjected to clustering using the DBScan algorithm, which creates clusters based on the density of the data points. For the second method, the normalized read counts for all the samples were used to create a principal component analysis. I then plotted the first three components in a 3-dimensional plot and used the same DBscan algorithm to generate a list of clusters. The generated clusters are then related back to the co-expression network with each gene in a cluster analyzed for how central it is in the co-expression network. By selecting genes that are very highly connected as targets for knockout, the probability of detecting a phenotypic change in colonization. Three genes have been selected from a total of 50 identified using this approach for mutant construction. The first mutant has been constructed, and assays are underway to determine the effect on colonization.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Crutcher, F.K., Moran-Diez, M.E., Krieger, I. V., and Kenerley C.M. 2019. Effects on hyphal morphology and development by the putative copper radical oxidase glx in Trichoderma virens suggest a novel role as a cell wall associated enzyme. Fungal Genetics and Biology. 131. Article Number:103245.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Crutcher, F.K., and Kenerley, C.M. 2019. Analysis of a putative glycosylation site in the Trichoderma virens elicitor SM1 reveals no role in protein dimerization. Biochemical and Biophysical Research Communications. 509:817-821.
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