Colletotrichum acutatum, the cause of strawberry anthracnose, is one of the most



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ABSTRACT
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 (19ºC and 25ºC). Mortality

of young seedlings was observed 4 days after inoculation with both species, reaching

50% within 10 days, using both techniques, at 25ºC. 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.


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