An Investigation of Two Modes of Plant Protection by the Biocontrol Agent Trichoderma virens
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The biocontrol fungus Trichoderma virens is an avirulent symbiont with the ability to control plant disease by the production of antibiotic compounds, induction of plant resistance to pathogens, and mycoparasitism of other fungi. In this document, the analysis of a putative terpene biosynthesis gene cluster (vir cluster) in T. virens is described. The vir cluster contains genes coding for four putative cytochrome P450s, an oxidoreductase, MFS transporter, and a terpene cyclase. To determine the function of this cluster in secondary metabolism biosynthesis, a strain of T. virens with a deletion of the putative cyclase, vir4, was constructed. Deletion mutants were deficient in the synthesis of sesquiterpene volatiles and complementation of vir4 restored this loss in transformants, albeit at a lower level of production. An analysis of phenotypic characteristics between mutant and wild-type strains did not identify any differences when the strain interacted with other fungi, bacteria, or Arabidopsis seedlings. Paralogs of the gene encoding the elicitor SM1 were examined as genetic sources for potential elicitors to induce systemic resistance in plants. A search of the T. virens genome revealed the presence of three paralogs of sm1. One paralog, sm3, was found to be expressed when grown in association with plant roots and in still-culture. The Pichia pastoris protein expression system was used to generate sufficient quantities of SM3 to allow characterization of its function. The purified protein from the yeast system (picSM3) was shown to be glycosylated and to increase expression of a plant defense gene in maize seedlings. Mutant strains in which sm3 was either deleted or over-expressed were constructed to further explore the potential of sm3 as an elicitor of ISR. The differential production of SM1 and SM3 by these strains suggested that SM1 and SM3 may be co-regulated and native SM3 may be glycosylated. To further understand the role of a putative glycosylation site as a mechanism to prevent dimerization and subsequent elicitor activity, a point mutation was created in a sm1 deletion strain. Analysis of the behavior of the protein demonstrates that the putative glycosylation site is not involved in protein aggregation and deletion of this site does not prevent the protein from testing positive for glycosylation. We propose that SM1is not glycosylated but instead may interact with an oligosaccharide or other small molecule. However, the mechanism of dimerization in SM1 remains unknown.
Crutcher, Frankie (2011). An Investigation of Two Modes of Plant Protection by the Biocontrol Agent Trichoderma virens. Doctoral dissertation, Texas A&M University. Available electronically from