טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRayzner Adi
SubjectInvolvement of the Enzyme DHS and the Translation
Initiation Factor eIF5A in Programmed Cell Death
and the Relation of these Proteins to
Resistance to Sigatoka ...
DepartmentDepartment of Biology
Supervisors Professor Emeritus Shimon Gepstein
Professor Benjamin Horwitz
Dr. Eli Khayat
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Most known land races of banana are susceptible to Black Leaf Streak Disease (BLSD).  The fungal pathogen Mycosphaerella fijiensis Morelet (MF) causes BLSD.  The disease is an epidemic in most of the world’s tropical banana growing countries. One major defense mechanism against pathogens is the hypersensitive response, in which reinforcement of cell walls and programmed death of plant cells set up a barrier limiting pathogen invasion. This response, however, is not effective against necrotrophic pathogens, which thrive on dead plant tissue.  In light of the parthenocarpic nature and triploidy, most edible banana cultivars are sterile. However, the genus Musa portrays a wealth of alleles that govern resistance traits to BLSD.  While triploid cultivars are useless to breeders due to the high occurrence of female sterility, several diploid cultivars have been shown to express a high level of resistance to the disease The initial defense response of most cultivars relies on programmed cell death. Being a hemi-biotroph, the fungus switches from a biotrophic to a necrotrophic life cycle. The transition to the necrotrophic phase is far more damaging to the host then the biotrophic phase. Aside from the ability of the necrotroph to exploit dead tissue, dead or dying plant cells can offer no effective defense against the pathogen. We have taken a transgenic approach that mimics the natural resistance of the land races. The transgenic approach is based on stopping or reducing the hypersensitive response, thus preventing the fungus from shifting to its necrotrophic phase. The goal is to slow down the progression of the fungus.

We have identified and cloned two genes from Musa that contribute to the process of programmed cell death. The translation initiation factor 5A (eIF5A) is activated by two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). We have isolated and cloned the genes encoding DHS and eight different isoforms of eIF5A from Cavendish banana. Based on homology comparison, we concluded that isoforms Ba1 and Ba2 are pathogenesis related.

In order to test our working hypothesis that inhibition of programmed cell death should slow the progress of BSLD, we used a specific inhibitor of DHS, GC7. The results demonstrated that the progression of the disease is significantly delayed. Subsequently, we prepared transgenic plants, expressing various RNA silencing constructs of the Ba1 and Ba2 isoforms of eIF5A, as well as DHS. In addition, we over-expressed the eIF5A orthologue from Populus eltoides (eastern cottonwood). The constructs were expressed either under constitutive or temporal promoters, all isolated from the Cavendish banana genome.

We conclude that silencing DHS or eIF5A is a useful approach to slow down, but not completely block Mycosphaerella fijiensis. The results indicate that regardless of the mechanism, stopping programmed cell death is effective in slowing down the progression of the disease. The effect eIF5A/DHS silencing in the host on the transition of the pathogen from the biotrophic to the necrotrophic stage needs further investigation. The results of both the transgenic strategies developed in this study have provided proof of concept for new environmentally-friendly approaches to fight the BSLD epidemic caused by Mycosphaerella fijiensis.