| Sclerotinia sclerotiorum infects over 400 plant species. The pathogen employs several mechanisms to overcome plant defenses including the production of oxalic acid which suppresses the hypersensitive response.It also utilizes hydrolytic enzymes to degrade cell structure and necrosis-inducing molecules to cause cell death. The work described in this thesis: 1) identified and elucidated the role of select proteins and enzymes in S. sclerotiorum pathogenesis, 2) determined how their respective genes were regulated throughout the different stages of the infection, and 3) examined their interaction with plant defenses.;Beneath the cuticle, host integrity is maintained by layers of polysaccharides in the plant cell wall. S. sclerotiorum produces a suite of pectin degrading enzymes, and a gene encoding a novel endopolygalacturonase (PG), SsPg2, is described. While SsPg2 was not expressed during infection of B. napus, the expression pattern of SsPg1indicated that it was involved in infection initiation and lesion expansion. SsPg1expression was regulated by a complex network involving feedback inhibition by galacturonic acid, ambient pH, and carbon availability. Two S. sclerotiorum PGs were demonstrated to induce necrosis in the host. This activity was inhibited by the B. napus polygalacturonase inhibitor proteins BnPGIP1 and BnPGIP2; however, expression of a single BnPGIP in transgenic plants was insufficient to provide practical resistance.;This study also identified two S. sclerotiorum Necrosis and Ethylene-inducing Peptides (NEPs) that caused necrotic lesions to form in host plants. SsNep2 was expressed at a higher level than SsNep1 during infection of B. napus and in both the necrotic zone and the leading margin, indicating that it is involved in lesion expansion and the later necrotic stage.;The signaling mechanisms that are involved in regulating the expression of these pathogenicity-related genes were examined, these included cAMP, Ca + flux, calcineurin and mitogen activated protein kinases (MAPKs). Disruption of the Smk3, the orthologue of the Slt cell wall integrity MAPK gene from Saccharomyces cerevisiae, led to loss of virulence on intact B. napus leaves, but not on leaves stripped of cuticle. This phenotype appeared to be associated with alterred mycelial polarity, branching and hydrophobicity and inability to form infection cushions.;The plant surface is covered by a waxy layer referred to as the cuticle. A gene encoding a cutinase was identified in the S. sclerotiorum genome. SsCutA was expressed after mycelia were placed in contact with the cuticle or other solid surfaces. Expression was limited to the very early stages of the infection suggesting that the enzyme only plays a role in cuticle penetration.;In summary, this study examined the integration and coordination of multiple S. sclerotiorium pathogencity factors during the infection and their interaction with the host. |
