Supervisors:  Yarden Oded, Freeman Stanley

Colletotrichum acutatum, the cause of strawberry anthracnose, is one of the most important fungal pathogens of this crop worldwide. However, the molecular basis of the infection process and pathogenicity components involved in the establishment of host plant colonization is poorly understood. Disruption of genes that result in the reduction or complete loss of disease symptoms by generation and screening for nonpathogenic mutants is a powerful tool for identifying pathogenicity-related genes. Successful application of this technique for plant fungal pathosystems requires reliable and rapid screening procedures. Development of a rapid in vitro bioassay in this study, enabled large scale screening and isolation of nonpathogenic mutants of Colletotrichum acutatum and C. gloeosporioides on strawberry seedlings. Inoculation was carried out on strawberry seedlings at two different developmental stages: 12-week-old (young) and 15-week-old (older) seedlings. A comparison was made between two inoculation techniques; (i) foliar dip and (ii) root soak, at two incubation temperatures (19�C and 25�C). Mortality of young seedlings was observed 4 days after inoculation with both species, reaching 50% within 10 days, using both techniques, at 25�C. However, mortality of older seedlings was delayed by 4 days compared to that in the young seedlings when using the root soak method. Disease development decreased in young and older seedlings at the lower temperature. This method, utilized for screening for non-pathogenic mutants, was also reliable in determining pathogenicity of the cucurbit-specific C. magna that did not cause disease symptoms on strawberry by either inoculation method. A random mutagenesis technique termed restriction-enzyme mediated integration (REMI) was used in order to identify genes that are important for colonization of strawberry. Over 1000 randomly mutated REMI strains were produced and screened for pathogenicity by the proposed method, resulting in a selection of five reducedvirulence isolates. Initial characterization of the reduced mutants included morphological characterization, molecular characterization of the insertion event and isolation of the flanking regions disrupted by the insertion vector. One of the five reduced pathogenicity mutants, designated Ca5, was the focus of this research. This mutant exhibited epiphytic hyphal growth and did not cause lesions on strawberry plants but grew necrotrophically when inoculated directly onto wounded stolons. In the absence of an external nitrogen source, the mutant exhibited extended germ-tube growth prior to appressorium formation. The deduced product of the impaired gene (nir1) is a C6 zinc-finger protein which is similar to NirA, an Aspergillus nidulans transcriptional regulator of nitrogen metabolism. Based on the molecular characterization of the Ca5 strain, the perception of nitrogen deprivation as a signal for induction of pathogenicity related morphogenesis development was studied. Inoculation of leaves with wild type (w.t) or non-pathogenic Ca5 conidia in the presence of a preferred nitrogen source resulted in massive epiphytic hyphal production, appressorium formation and rapid symptom development. In an attempt to determine nitrogen availability during the infection process, the expression of nitrate reductase (nit1) and glutamine synthetase (gln1) was investigated. Expression of C. acutatum w.t nit1 and gln1 was induced by nitrate but only nit1 expression was repressed while growing in complete nutrient supply. Furthermore, nit1 transcription increased during the appressorium-production stage, indicating that nitrogen starvation constitutes a cue for the regulation of appressorium development. Presence of the nit1 transcript during various phases of infection is indicative of partial nitrogen starvation in planta. cAMP-dependent protein kinase A (PKA) was determined to be a negative regulator of immediate post-germination appressoria formation in the w.t. Since inhibition of PKA activity in the nir1 mutant did not affect appressoria formation, it is suggested that NIR1 acts either in parallel or downstream of the PKA pathway. The current results show that nir1 is a pathogenicity determinant and a regulator of preinfection development under nitrogen-starvation conditions and that nitrogen availability is a significant factor in the pre-penetration phase. Given that the nir1 gene plays a role in a nitrogen-regulated signaling pathway and that C. acutatum pathogenicity can be attributed to its ability to sense and respond to nitrogen limitation, the relevance of nutrient regulation of morphogenesis for the pathogenicity of C. acutatum was further studied by performing a proteomicbased approach. To determine whether nitrogen starvation constitutes a cue for regulating developmental processes and pathogenicity, the cellular outcome of changes to nitrogen deprivation, in context to development and early stages of pathogenicity, was investigated. Quantitative analysis of proteins synthesized during appressoria formation, under nitrogen limiting conditions or complete nutrient supply, compared to the Ca5 non-pathogenic mutant, revealed significant changes in the abundance of different proteins. The function of the proteins that were up-regulated or down-regulated can be grouped into the following main categories: energy metabolism, nitrogen and amino acid metabolism, protein synthesis and degradation, response to stress and reactive oxygen scavengers. Differential display analysis of proteins involved in growth under limiting conditions and during appressoria formation could be useful to further understand changes leading to establishment of the early infection stage in additional host pathogen interactions. Proteins patterns of different developmental stages and growth conditions revealed variation in abundance of proteins belonging to the ROS (Reactive oxygen species) scavenger machinery. Members belonging to the ROS scavenger machinery, CuZn superoxide dismutase (SOD) and glutathione peroxidase (GPX) were upregulated at the appressoria formation stage and nitrogen limiting conditions, compared to growth in complete nutrient supply, whereas the bifunctional catalase (CAT2) was predominantly detected at the appressoria stage. To follow changes of ROS levels during fungal development, qualitative and quantitative ROS production was measured in response to different nutritional conditions. In vitro fungal ROS generation was markedly increased under nitrogen limiting conditions, a precondition for appressorium formation, compared to growth in complete nutrient supply. Plant ROS accumulation in vivo was detected at the stolon appoplastic space, exclusively at sites of direct physical contact between appressoria structures and the host. Fungal ROS production within germinating conidia during the pre-penetration phase and over the course of penetration and colonization was prominent. The role of ROS as a regulator of appressoria development was investigated using a pharmacological based approach. Application of an exogenous antioxidant in vitro, such as N-acetyl-Lcysteine or mannitol, reduced ROS production as well as appressorium formation. These results suggest that ROS production is an early fungal response to nitrogen starvation, modulating initial stages of pathogen development. A general up-regulation in metabolic activity during appressoria formation and nutrient deficiency conditions was observed via the proteomic-based analysis. Abundance of proteins belonging to the glyoxylate cycle and lipid metabolism, such as malate dehydrogenase (MDH), formate dehydrogenase (FDH) and acetyl CoA acetyltransferase was observed during the course of appressoria production. In contrast, down-regulation of isocitrate dehydrogenase (IDH), detected during appressoria formation may indicate that the organism is preparing for lipid-based energy supply. The present study demonstrates that the developmental processes CuZn superoxide dismutase (SOD) and glutathione peroxidase (GPX) were upregulated at the appressoria formation stage and nitrogen limiting conditions, compared to growth in complete nutrient supply, whereas the bifunctional catalase (CAT2) was predominantly detected at the appressoria stage. To follow changes of ROS levels during fungal development, qualitative and quantitative ROS production was measured in response to different nutritional conditions. In vitro fungal ROS generation was markedly increased under nitrogen limiting conditions, a precondition for appressorium formation, compared to growth in complete nutrient supply. Plant ROS accumulation in vivo was detected at the stolon appoplastic space, exclusively at sites of direct physical contact between appressoria structures and the host. Fungal ROS production within germinating conidia during the pre-penetration phase and over the course of penetration and colonization was prominent. The role of ROS as a regulator of appressoria development was investigated using a pharmacological based approach. Application of an exogenous antioxidant in vitro, such as N-acetyl-Lcysteine or mannitol, reduced ROS production as well as appressorium formation. These results suggest that ROS production is an early fungal response to nitrogen starvation, modulating initial stages of pathogen development. A general up-regulation in metabolic activity during appressoria formation and nutrient deficiency conditions was observed via the proteomic-based analysis. Abundance of proteins belonging to the glyoxylate cycle and lipid metabolism, such as malate dehydrogenase (MDH), formate dehydrogenase (FDH) and acetyl CoA acetyltransferase was observed during the course of appressoria production. In contrast, down-regulation of isocitrate dehydrogenase (IDH), detected during appressoria formation may indicate that the organism is preparing for lipid-based energy supply. The present study demonstrates that the developmental processes occurring under nutritional deprivation are facilitated by metabolic shifts which may be mediated by ROS production. Regarding the nir1 mutation, findings pertaining to the Ca5 non-pathogenic mutant are intriguing and represent the initial steps in defining the factors controlling virulence in C. acutatum infecting strawberry at the molecular level. This study utilizing the Ca5 mutant demonstrates that many diverse physiological and morphogenetic processes may be classified as being mediated by pathogenicity genes and emphasize the perception of nitrogen starvation regulation on development and pathogenicity in C. acutatum.