טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentEisa Jamela
SubjectUnderstanding the Role of PH in Colonization of Plant
Roots by Trichoderma Virens
DepartmentDepartment of Biology
Supervisor Dr. Benjamin Horwitz
Full Thesis textFull thesis text - English Version


Abstract

Environmental pH is an important environmental cue affecting Trichoderma virens development and gene expression. Trichoderma virens is known for its potential to be used as a biocontrol agent to suppress plant pathogens and promote plant growth. The Trichoderma virens-plant roots interaction depends on a cross-talk in the apoplast, which is the region occupied by plant cell walls, encountered by the fungus as it invades the root. Identifying the major players during this interaction is of great importance. Interestingly, several studies have shown that the pH shift in the host tissue plays a vital role during colonization of plants by fungi, especially the pathogenic ones, since it regulates the penetration and the infectious potential of the fungus. However, the involvement of pH in the symbiotic fungi Trichoderma virens-plant interaction is not well understood. Elucidating its role during this interaction at the molecular level will allow developing genetically engineered strains with superior biocontrol potential. The aim of this study was to elucidate the role of pH in colonization of plant roots by Trichoderma virens, both at the microscopic and macroscopic levels. This was pursued by working with two mutants in genes that we suggest are involved in the colonization mechanism: one is a pma1 mutant, with deletion of a plasma membrane ATPase; we also worked with a PacC mutant, with an acid mimicking phenotype which behaves as if it is in an acidic environment and expresses acid expressed genes. At the macroscopic level we determined the extent of colonization by the wild type (WT), and deletion mutants in PacC and Pma1, by assays of colony forming units (CFU) and by confocal microscopy. At the microscopic level, we evaluated  the changes in the apoplastic pH during maize roots-T.virens interaction using 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) staining. A qPCR method was also used to evaluate colonization by T. virens. Furthermore, we determined whether the light signal is important for changing the local environmental pH by Trichoderma virens. Our findings indicate that qPCR method is a suitable quantitative assay to evaluate the colonization of maize roots by Trichoderma virens strains. In addition, a fluorescent dye can be used to track the pH changes in the apoplast during Trichoderma virens- maize interaction. PacC and pma1 mutants show increased colonization compared to the WT, both at the microscopic level suggesting that these genes are not essential for the colonization mechanism and loss of them may improve the colonization efficacy. In addition, we found that the light signal is needed for alkalization of the medium by the WT and PacC mutant.